3D Co-culture Research Articles

LINC01119 encapsulated by cancer-associated adipocytes-derived exosomes promotes M2 polarization of macrophages to induce immune escape in ovarian cancer in a 3D co-culture cell-based model.

In the present study, we sought to clarify the role of LINC01119 delivered by cancer-associated adipocytes (CAAs)-derived exosomes (CAA-Exo) and its mechanistic actions in ovarian cancer (OC). The expression of LINC01119 was determined in OC, and the relationship between LINC01119 expression and the prognosis of OC patients was analyzed. Besides, 3D co-culture cell models were constructed using green fluorescent protein-labeled OC cells and red fluorescent protein-labeled mature adipocytes. Mature adipocytes were co-cultured with OC cells to induce CAA. Macrophages treated with CAA-Exo were co-cultured with SKOV3 cells following ectopic expression and depletion experiments of LINC01119 and SOCS5 to detect M2 polarization of macrophages, PD-L1 level, proliferation of CD3+ T cells, and cytotoxicity of T cells to SKOV3 cells. LINC01119 was elevated in the plasma Exo of OC patients, which was related to shorter overall survival in OC patients. LINC01119 expression was increased in CAA-Exo, which could upregulate SOCS5 in OC. Finally, CAA-Exo carrying LINC01119 induced M2 polarization of macrophages to promote immune escape in OC, as evidenced by inhibited CD3+ T cell proliferation, increased PD-L1 level, and attenuated T cell toxicity to SKOV3 cells. In conclusion, the key findings of the current study demonstrated the promoting effects of CAA-Exo containing LINC01119 mediating SOCS5 on M2 polarization of macrophages and immune escape in OC.

3D co-culture models demonstrate radiation-mediated activation of tumor-associated macrophages

Abstract Type I IFN signaling mediated by STING pathway is important for the induction of radiation-stimulated adaptive immune responses in responsive tumors. Macrophages recruited to tumors following radiotherapy (RT) can influence the response to RT. Reprogramming immunosuppressive M2-like macrophages in tumors to exhibit a more activated M1-like phenotype can help improve response to therapies. Here, we hypothesize that a 3D co-culture model of cancer cells and macrophages will be more contextually relevant to the events occurring in tumors and can be used to study the crosstalk that occurs between these cells in vivo. We initially optimized seeding densities of murine MC38 colorectal carcinoma cells to develop spheroids of consistent size. Using GFP+ macrophages and Hoechst stained MC38, we imaged the development and localization of these cells during spheroid formation. Next, co-culture spheroids were exposed to 12 Gy radiation on day 1. Viability and phenotype of macrophages in spheroid co-cultures was assessed on day 4 using flow cytometry by staining for CD80, CD86, MHC Class II, CD69 and CD206 expression. STING ligand, cyclic di-AMP (CDA) treated spheroid co-cultures were used as positive controls. CDA treatment significantly increased CD80/86 expression in 3D co-cultures. Treatment with 12 Gy radiation showed similar effect. Interestingly, macrophages derived from MyD88 Lyz2Cre, MyD88 fl/fland STING knockout mice also showed a significant increase in CD80/86 and CD40, suggesting that macrophage activation was independent of MyD88 or STING signaling. Ongoing studies aim to explore the pathways activated on irradiation in these cells and further cross-validate with the macrophage phenotypes observed in vivo in response to RT.

Hypoxia promotes inflammatory fibroblast formation in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy characterized by poor response to all existing therapies. Although immunotherapy has shown great promise against multiple deadly cancers, it has been largely ineffective in PDAC. This lack of response is in part attributed to its extensive, fibroinflammatory stroma and hypoxic microenvironment. Cancer-associated fibroblasts (CAFs) are the predominant stromal cell type in PDAC, and single-cell transcriptomics of human and mouse PDAC has recently revealed a distinct CAF subpopulation, inflammatory CAFs (iCAFs). These inflammatory fibroblasts produce high levels of cytokines and chemokines in PDAC, and have the potential to contribute to its immunosuppressive microenvironment and tumorigenesis. By injecting a hypoxia probe into PDAC mouse models, we recently found that iCAFs predominantly reside in hypoxic tumor regions. We also observed that the hypoxia-related gene signature is positively enriched in iCAFs in human PDAC samples. Importantly, by exposing three-dimensional (3D) co-cultures of pancreatic cancer cells and fibroblasts to either hypoxia or normoxia, we showed that hypoxia induces IL1α from cancer cells and that IL1α is required for hypoxia-mediated iCAF formation. Our data implicate hypoxia as a critical regulator of tumor stroma. Efforts are ongoing to understand the role of hypoxia in the crosstalk between cancer cells, fibroblasts, and immune cells in driving an immunosuppressive tumor microenvironment. Supported by a grant from the NIH (T32 AI007413) and the University of Michigan Rackham Merit Fellowship.

Activation of the integrated stress response (ISR) pathways in response to Ref-1 inhibition in human pancreatic cancer and its tumor microenvironment.

Pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC) is characterized by a profound inflammatory tumor microenvironment (TME) with high heterogeneity, metastatic propensity, and extreme hypoxia. The integrated stress response (ISR) pathway features a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) and regulate translation in response to diverse stress conditions, including hypoxia. We previously demonstrated that eIF2 signaling pathways were profoundly affected in response to Redox factor-1 (Ref-1) knockdown in human PDAC cells. Ref-1 is a dual function enzyme with activities of DNA repair and redox signaling, responds to cellular stress, and regulates survival pathways. The redox function of Ref-1 directly regulates multiple transcription factors including HIF-1α, STAT3, and NF-κB, which are highly active in the PDAC TME. However, the mechanistic details of the crosstalk between Ref-1 redox signaling and activation of ISR pathways are unclear. Following Ref-1 knockdown, induction of ISR was observed under normoxic conditions, while hypoxic conditions were sufficient to activate ISR irrespective of Ref-1 levels. Inhibition of Ref-1 redox activity increased expression of p-eIF2 and ATF4 transcriptional activity in a concentration-dependent manner in multiple human PDAC cell lines, and the effect on eIF2 phosphorylation was PERK-dependent. Treatment with PERK inhibitor, AMG-44 at high concentrations resulted in activation of the alternative ISR kinase, GCN2 and induced levels of p-eIF2 and ATF4 in both tumor cells and cancer-associated fibroblasts (CAFs). Combination treatment with inhibitors of Ref-1 and PERK enhanced cell killing effects in both human pancreatic cancer lines and CAFs in 3D co-culture, but only at high doses of PERK inhibitors. This effect was completely abrogated when Ref-1 inhibitors were used in combination with GCN2 inhibitor, GCN2iB. We demonstrate that targeting of Ref-1 redox signaling activates the ISR in multiple PDAC lines and that this activation of ISR is critical for inhibition of the growth of co-culture spheroids. Combination effects were only observed in physiologically relevant 3D co-cultures, suggesting that the model system utilized can greatly affect the outcome of these targeted agents. Inhibition of Ref-1 signaling induces cell death through ISR signaling pathways, and combination of Ref-1 redox signaling blockade with ISR activation could be a novel therapeutic strategy for PDAC treatment.

Organotypic 3D Co-Culture of Human Pleura as a Novel In Vitro Model of Staphylococcus aureus Infection and Biofilm Development.

Bacterial pleural infections are associated with high mortality. Treatment is complicated due to biofilm formation. A common causative pathogen is Staphylococcus aureus (S. aureus). Since it is distinctly human-specific, rodent models do not provide adequate conditions for research. The purpose of this study was to examine the effects of S. aureus infection on human pleural mesothelial cells using a recently established 3D organotypic co-culture model of pleura derived from human specimens. After infection of our model with S. aureus, samples were harvested at defined time points. Histological analysis and immunostaining for tight junction proteins (c-Jun, VE-cadherin, and ZO-1) were performed, demonstrating changes comparable to in vivo empyema. The measurement of secreted cytokine levels (TNF-α, MCP-1, and IL-1β) proved host-pathogen interactions in our model. Similarly, mesothelial cells produced VEGF on in vivo levels. These findings were contrasted by vital, unimpaired cells in a sterile control model. We were able to establish a 3D organotypic in vitro co-culture model of human pleura infected with S. aureus resulting in the formation of biofilm, including host-pathogen interactions. This novel model could be a useful microenvironment tool for in vitro studies on biofilm in pleural empyema.

Common fragrance chemicals activate dendritic cells in coculture with keratinocytes.

Fragrances are important contact allergens; however, investigation of their skin sensitization potency has been challenging in new approach methods (NAMs). Many fragrance chemicals are susceptible to autoxidation or can be metabolized by enzymes constitutively expressed in skin keratinocytes. Strong sensitizers can be formed in both of these processes. Further, keratinocytes can modulate the dendritic cell (DC) activation and maturation potential, a key event in the acquisition of contact allergy. To evaluate the 2D coculture model consisting of keratinocytes and DCs using different weak to moderate sensitizing fragrance chemicals. Further, to investigate fragrances and related oxidation products in the in vitro model and compare to in vivo data. Chemicals were tested in the coculture activation test (COCAT), consisting of HaCaT keratinocytes and THP-1 cells. THP-1 cell surface expression of costimulatory and adhesion molecules (CD86 and CD54) collected after 24 h incubation with the chemicals was analysed using flow cytometry. Twenty-four molecules were tested positive, three were negative (n = 27). Four pairs were evaluated, with aldehydes showing a 6- to 13-fold stronger responses compared to their corresponding alcohols. Results provide insight into the activation of DC in their natural environment of keratinocytes. α,β-Unsaturated alcohols were classified as weaker sensitizers compared to their corresponding aldehydes. In sum, testing of fragrances retrieved results in good agreement with in vivo data.

G protein-coupled receptor 183 mediates the sensitization of Burkitt lymphoma tumors to CD47 immune checkpoint blockade by anti-CD20/PI3Kδi dual therapy.

Immunotherapy-based regimens have considerably improved the survival rate of B-cell non-Hodgkin lymphoma (B-NHL) patients in the last decades; however, most disease subtypes remain almost incurable. TG-1801, a bispecific antibody that targets CD47 selectively on CD19+ B-cells, is under clinical evaluation in relapsed/refractory (R/R) B-NHL patients either as a single-agent or in combination with ublituximab, a new generation CD20 antibody. A set of eight B-NHL cell lines and primary samples were cultured in vitro in the presence of bone marrow-derived stromal cells, M2-polarized primary macrophages, and primary circulating PBMCs as a source of effector cells. Cell response to TG-1801 alone or combined with the U2 regimen associating ublituximab to the PI3Kδ inhibitor umbralisib, was analyzed by proliferation assay, western blot, transcriptomic analysis (qPCR array and RNA sequencing followed by gene set enrichment analysis) and/or quantification of antibody-dependent cell death (ADCC) and antibody-dependent cell phagocytosis (ADCP). CRISPR-Cas9 gene edition was used to selectively abrogate GPR183 gene expression in B-NHL cells. In vivo, drug efficacy was determined in immunodeficient (NSG mice) or immune-competent (chicken embryo chorioallantoic membrane (CAM)) B-NHL xenograft models. Using a panel of B-NHL co-cultures, we show that TG-1801, by disrupting the CD47-SIRPα axis, potentiates anti-CD20-mediated ADCC and ADCP. This led to a remarkable and durable antitumor effect of the triplet therapy composed by TG-1801 and U2 regimen, in vitro, as well as in mice and CAM xenograft models of B-NHL. Transcriptomic analysis also uncovered the upregulation of the G protein-coupled and inflammatory receptor, GPR183, as a crucial event associated with the efficacy of the triplet combination. Genetic depletion and pharmacological inhibition of GPR183 impaired ADCP initiation, cytoskeleton remodeling and cell migration in 2D and 3D spheroid B-NHL co-cultures, and disrupted macrophage-mediated control of tumor growth in B-NHL CAM xenografts. Altogether, our results support a crucial role for GPR183 in the recognition and elimination of malignant B cells upon concomitant targeting of CD20, CD47 and PI3Kδ, and warrant further clinical evaluation of this triplet regimen in B-NHL.

Metabolic profile of mesenchymal stromal cells and macrophages in the presence of polyethylene particles in a 3D model

BackgroundContinuous cross talk between MSCs and macrophages is integral to acute and chronic inflammation resulting from contaminated polyethylene particles (cPE); however, the effect of this inflammatory microenvironment on mitochondrial metabolism has not been fully elucidated. We hypothesized that (a) exposure to cPE leads to impaired mitochondrial metabolism and glycolytic reprogramming and (b) macrophages play a key role in this pathway.MethodsWe cultured MSCs with/without uncommitted M0 macrophages, with/without cPE in 3-dimensional gelatin methacrylate (3D GelMA) constructs/scaffolds. We evaluated mitochondrial function (membrane potential and reactive oxygen species—ROS production), metabolic pathways for adenosine triphosphate (ATP) production (glycolysis or oxidative phosphorylation) and response to stress mechanisms. We also studied macrophage polarization toward the pro-inflammatory M1 or the anti-inflammatory M2 phenotype and the osteogenic differentiation of MSCs.ResultsExposure to cPE impaired mitochondrial metabolism of MSCs; addition of M0 macrophages restored healthy mitochondrial function. Macrophages exposed to cPE-induced glycolytic reprogramming, but also initiated a response to this stress to restore mitochondrial biogenesis and homeostatic oxidative phosphorylation. Uncommitted M0 macrophages in coculture with MSC polarized to both M1 and M2 phenotypes. Osteogenesis was comparable among groups after 21 days.ConclusionThis work confirmed that cPE exposure triggers impaired mitochondrial metabolism and glycolytic reprogramming in a 3D coculture model of MSCs and macrophages and demonstrated that macrophages cocultured with MSCs undergo metabolic changes to maintain energy production and restore homeostatic metabolism.

The characterization of the sensitive ovarian cancer cell lines A2780 and W1 in response to ovarian CAFs

PurposeThe cancer-associated fibroblasts (CAFs) are one of the most abundant components of the tumor microenvironment (TME). CAFs have been implicated in tumor progression, extracellular matrix (ECM) remodeling, and treatment resistance. Drug resistance is the primary limiting factor in achieving cures for patients with cancer, particularly ovarian cancer. Therefore, inhibiting CAFs can be an effective strategies for cancer treatment. In this research, we studied whether CAFs have an influence on drug-sensitive ovarian cancer cells to become more resistant. We examined the influence of CAFs on genes and proteins expression changes in sensitive ovarian cancer cells. We prepared a 3D co-culture to investigate the role of CAFs on cancer cell morphology. MethodsHere, we performed a detailed analysis of drug-sensitive ovarian cancer cell lines (A2780 and W1) and the influence of ovarian CAFs on the A2780 and W1 cells morphology, genes and proteins expression. The 2D and 3D cultures, genes expression analysis (TaqMan qPCR), and proteins expression (Western blot analysis) were assessed in this study. ResultsWe observed upregulation of ABCC5, CYP2C8, CYP2C9, and DHFR mRNA in cell lines supplemented by CAFs medium. We showed fibronectin overexpression and COL3A1 downregulation after supplementation with CAFs. Co-culturing with CAFs prevented the formation of spheroids in 3D conditions. ConclusionWe demonstrated that the process of drug resistance in ovarian cancer cells is launched by CAFs. CAFs not only simulate cancer cells to produce drug transporters and specific enzymes production, but also remodel the TME to increase drug resistance. We believe that cancer progression and migration is due to the CAFs po-tumorigenic activity.

Proteolytically degradable PEG hydrogel matrix mimicking tumor immune microenvironment for 3D co-culture of lung adenocarcinoma cells and macrophages

Tumor-associated macrophages and monocytes are the major stromal cell types found in the tumor immune microenvironment (TIME), which modulates tumor progression, invasion, and chemoresistance. To address the need for an in vitro three-dimensional tumor model for understanding the complex cellular interactions within the TIME, we propose a TIME-mimetic co-culture matrix composed of photo-crosslinked poly(ethylene glycol) hydrogels mimicking the characteristics of the tumor and stroma. Desmoplasia-mimetic microgels encapsulating lung adenocarcinoma cells (A549) were embedded with monocyte- or macrophage-type U937 cells in normal stroma-mimetic hydrogel, increasing the proximity between the two cell types. By modulating the proteolytic degradability of the hydrogels, we could separate different cell types with high purities for use in orthogonal assays. In addition, we demonstrated that U937 cells had distinct influences on A549 cell death depending on their activation states (i.e. monocyte, M0, or M1 phenotype). M1 macrophages suppressed tumor growth and increased the susceptibility of A549 cells to cisplatin. In contrast, monocytes upregulated cancer stem cell markers (OCT4, SOX2, and SHH) of A549 cells, showing M2-like features, such as downregulated expression of proinflammatory markers (IL6 and TNFα). These findings suggest that this co-culture system is potentially used for investigation of heterotypic cellular interactions in the TIME.

Exploitation of In Vivo-Emulated In Vitro System in Advanced Food Science.

Reasonable model construction contributes to the accuracy of experimental results. Multiple in vivo models offer reliable choices for effective evaluation, whereas their applications are hampered due to adverse features including high time-consumption, high cost and ethical contradictions. In vivo-emulated in vitro systems (IVE systems) have experienced rapid development and have been brought into food science for about two decades. IVE systems' flexibly gathers the strengths of in vitro and in vivo models into one, reflecting the results in an efficient, systematic and interacted manner. In this review, we comprehensively reviewed the current research progress of IVE systems based on the literature published in the recent two decades. By categorizing the IVE systems into 2D coculture models, spheroids and organoids, their applications were systematically summarized and typically exemplified. The pros and cons of IVE systems were also thoroughly discussed, drawing attention to present challenges and inspiring potential orientation and future perspectives. The wide applicability and multiple possibilities suggest IVE systems as an effective and persuasive platform in the future of advanced food science.

Shotgun Proteomics of Co-Cultured Leukemic and Bone Marrow Stromal Cells from Different Species as a Preliminary Approach to Detect Intercellular Protein Transfer

Cellular interactions within the bone marrow microenvironment modulate the properties of subsets of leukemic cells leading to the development of drug-resistant phenotypes. The intercellular transfer of proteins and organelles contributes to this process but the set of transferred proteins and their effects in the receiving cells remain unclear. This study aimed to detect the intercellular protein transfer from mouse bone marrow stromal cells (OP9 cell line) to human T-lymphoblasts (CCRF-CEM cell line) using nanoLC-MS/MS-based shotgun proteomics in a 3D co-culture system. After 24 h of co-culture, 1513 and 67 proteins from human and mouse origin, respectively, were identified in CCRF-CEM cells. The presence of mouse proteins in the human cell line, detected by analyzing the differences in amino acid sequences of orthologous peptides, was interpreted as the result of intercellular transfer. The transferred proteins might have contributed to the observed resistance to vincristine, methotrexate, and hydrogen peroxide in the co-cultured leukemic cells. Our results suggest that shotgun proteomic analyses of co-cultured cells from different species could be a simple option to get a preliminary survey of the proteins exchanged among interacting cells.

Abstract 201: Establishment of 3D co-culture spheroid model for in- vitro drug screening

Abstract Melanoma is an aggressive form of skin cancer that accounts for 75% of deaths of all skin cancers in the United States. Moreover, the number of cases is increasing at an alarming rate than any other cancer worldwide. But what is more alarming is the lack of chemo preventive or therapeutic drugs that are easily accessible to people and the absence of an efficient, effective and reliable model or platform for drug screening. A growing scientific and observational analyses suggests that antioxidants contain in blueberry inhibit tumor growth, and help slow down esophageal, lung, mouth, pharynx, endometrial, pancreatic, prostate, and colon cancers. Lately, the use of 3D multicellular spheroid culture has become a potential link to bridge the gap between monolayer culture and animal model. The treatment of blueberry extract (1mg) on the spheroids of A375 cells showed a disintegration of these spheroids and reduction in their growth. To optimize the culture conditions further herein, we used a novel three-dimensional (3D) coculture system with melanoma (A375), human fibroblast (HF-1) and Human umbilical endothelial cells (HUVECs) to study the effectiveness of drug in vitro. The multicellular nature of the coculture spheroid stimulates the microenvironment of the original tumor which makes it more efficient in drug screening process. The immunofluorescence assay on the 3D spheroid co-culture shows the interactions among these three cells which mimics the tumor microenvironment. We further analyzed the combination of blueberry extract and resveratrol which proved the effectiveness of blueberry extract as a potential chemosensitizer with resveratrol for skin cancer therapy. The complementary effect on the inhibition of A375 cell proliferation were observed when treated with 8 mg blueberry and 10 mM of resveratrol. The results of MTT assay indicates that the blueberry extracts showed potential cytotoxic activity against A375 cell line. 3D spheroid assay and 3D spheroid co-culture assays further confirmed the effectiveness of blueberry and resveratrol combination. Therefore, this platform could be applied for in vitro based drug screening studies. To conclude cytotoxic effects of the blueberry extract in human melanoma cells make them good candidates for further pharmacological studies to discover effective drugs for melanoma therapy. Citation Format: Christella J. Nelson, Vino T. Cheriyan, Abhishek Pandit, Joseph Francis, Alan Tackett. Establishment of 3D co-culture spheroid model for in- vitro drug screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 201.

Abstract 1328: Building a 3D co-culture model for metastatic pancreatic ductal cancer using Curiochips microphysiological system

Abstract Despite the use of multi-agent conventional chemotherapy regimens, metastatic pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal solid tumors with a 5-year survival of 9%. PDAC is characterized by a complex tumor microenvironment with multilayered interplay between neoplastic and stromal cells and the natural extracellular matrix (ECM). To date, models that recapitulate the complexity of these features are only partially reproduced ex vivo and lack the capacity to recreate PDAC tumor microenvironment physiology, fluid flow and the ECM architecture. To address these issues, we developed Curiochips MPS platform for metastatic PDAC on-a-chip. To replicate an accurate PDAC complex tumor microenvironment, we investigated human pancreatic cancer cell lines with/without SMAD4 mutation as a test model. SMAD4 is among the most frequently altered genes in PDAC and its mutation accelerates pancreatic cancer cell migration. To tailor cell-ECM interactions and recreate natural biochemical environment of tumors, we examined various ECM conditions. ECM substitutes such as type I collagen and fibrin gel offer mechanical properties to closely mimic the tumor ECM. We accessed the effect of ECM condition on the pancreatic stellate cells (PSC) activation and pancreatic cancer cell migration using PANC-1 (SMAD4-WT) and BxP3 (SMAD4-null) cell lines. Notably, type I collagen induced more PSC activation than fibrin gel. Interestingly BxP3 spheroids migrated toward to the endothelial compartment compared to PANC-1. Angiogenesis of endothelial cells was more prominent in co-culture with BxP3 than PANC-1. Furthermore, we are building the metastatic model using PDAC patient derived organoids and co-culture with primary PSC in different types of ECM. Preliminary analysis showed functional responses such as 3D organoid migration and aggregation, PSC activation and migration controlled by specific properties of EMC. Our data highlight the importance of the ECM composition on PDAC migration, PSC activation and angiogenesis in the context of genetic heterogeneity and presence/absence oncogenic mutation. The 3D co-culture platform, Curiochips offers suitable and tailored solution to investigate tumor-stroma interaction and potentially can be used as a platform for drug discovery and precision oncology to target PDAC desmoplastic tumor microenvironment. Citation Format: Sumin Kim, Yeongmin Choi, Jihye Baek, Bharti Garg, Jan Pencik, Andrew M. Lowy, Herve Tiriac, Sanghee Yoo. Building a 3D co-culture model for metastatic pancreatic ductal cancer using Curiochips microphysiological system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1328.

Abstract 3629: Calcium signalling and the breast cancer microenvironment

Abstract Calcium is a key regulator of a variety of pathways important in breast cancer progression, including those involved in cell proliferation and invasiveness. Calcium signaling is implicated in processes relevant to the breast cancer tumor microenvironment and altered calcium influx is a feature of cancer associated fibroblasts. In these studies, simultaneous use of genetically encoded calcium indicators with distinct fluorescence spectral properties (GCaMP6m or JRCaMP1b), were used to explore the cross-talk between breast cancer cells and fibroblasts via calcium signaling and changes in different cellular compartments. HMF3S human fibroblast cells co-cultured with breast cancer cells expressing GCaMP6m or JRCaMP1b were assessed in 2D and 3D co-culture using automated epi-fluorescence or confocal microscopy. Mitochondrial calcium levels were assessed using targeted GCaMP6m (2mtGCaMP6m) in HMF3S cells expressing JRCaMP1b. Results showed distinct spatial and temporal changes in free calcium levels between both fibroblasts and breast cancer cells and the mitochondria and cytosol during activation. These methodological advances provide an opportunity to better understand calcium signaling in cancer associated fibroblasts. These techniques will also help identify the calcium channels and pumps that could be targeted to manipulate the breast cancer tumor microenvironment to inhibit pathways important in breast cancer progression. Citation Format: Alice H. Bong, Krystyna A. Gieniec, Francisco Sadras, Mélanie Robitaille, Sarah J. Roberts-Thomson, Felicity M. Davis, Gregory R. Monteith. Calcium signalling and the breast cancer microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3629.

Abstract 4419: Use of luciferase-labeled target cells to explore immune cell killing in high throughput format in 2D and 3D co-cultures

Abstract With the development and approval of cutting-edge immune-based approaches for cancer treatment in recent years, the global immune-oncological (IO) pipelines have dramatically increased. To overcome the challenges of rising treatment resistance, new strategies are being developed pointing towards enhancing and sustaining T cell-mediated cytotoxicity, exploiting NK, monocytes and γδT cell killing capacity, and combinatorial therapies, among others. Examples of such strategies are the bispecific antibodies, which are mainly T-cell (BiTE) engagers, but also NK-cell engagers. In addition to the FDA approved immune checkpoint inhibitors, monoclonal antibodies against tumor antigens are being developed to enhance the cytotoxic response of NK and macrophages/monocytes through antibody-induced cell cytotoxicity (ADCC) To facilitate the efficacy analysis of BiTEs, we developed a 384-well format assay system based on luciferase-labeled tumor cells (advantages: high-throughput capability, non-radioactive, non-toxic, relatively low cost, and easily adaptable to automation). As only the tumor cells are labeled, the specific detection of luciferase exclusively correlates to the number of viable tumor cells, allowing for co-cultures with high excess of effector cells without the effector cells causing interference. Due to high sensitivity, few tumor cells are required for a sufficient signal/noise ratio, keeping the overall need for primary effector cells to a minimum, even at high effector/target cell ratios. This is of great advantage when working with rare cytotoxic subpopulations (e.g. NK cells), precious samples from patients, or simply saves resources when performing large exploratory studies. In addition, combination of low 384-well format volumes with nanodrop agent dispensing technology minimizes the amounts of expensive antibodies and other agents. Applying this technology, we evaluated the combinatorial effects of Blinatumomab with other clinically relevant agents on the luciferase-labeled GCB-like DLBCL cell line OCI-LY1. Depending on compound combination, we observe synergistic (e.g. Lenalidomide) but also antagonistic effects (e.g. MEK-inhibitor, Selumetinib, and MCL-1-inhibitor, S63845). We also developed CD3-stimulated T cell killer assays either using target cells grown in monolayer or spheroids, with the intent to study checkpoint inhibitors or other T cell activation modulators. In contrast to target cells grown in monolayers, immune cytotoxicity of spheroids by anti-CD3 stimulated T cells was incomplete, showing the additional challenge for effector cells when using 3D cultures as targets. Moreover, we show an ADCC example using NK as cytotoxic cells. Our data supports the outstanding usefulness of this methodological approach for the exploration of the overall efficiency of a therapy to enhance tumor cell killing by immune effector cells. Citation Format: Carla Castro, Philipp Metzger, Daniel Feger, Sarah Ulrich, Oliver Siedentopf, Jan E. Ehlert, Holger Weber. Use of luciferase-labeled target cells to explore immune cell killing in high throughput format in 2D and 3D co-cultures. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4419.

Abstract 4559: Evaluation of the chemotherapy potential to improve the anti-cancer efficacy of PD-L1 inhibition using novel 3D microfluidic co-culture platform

Abstract Immunotherapy is now recognized as a powerful therapeutic approach to treat and cure cancer. However, the objective response rates for most solid tumors are low (below 30%). Accumulating evidence has shown that chemotherapy can increase the efficacy of immune checkpoint blockade and improve cancer outcomes. We have investigated if chemotherapy can modulate anti-PDL1 enhanced T cell cytotoxicity in breast cancer using novel in vitro 3D co-culture assay system. 3D co-culture of cancer and immune cells is a powerful platform for disease modeling and therapies testing because it can mimic the tumor micro-environment and complex cellular interactions. In this study we evaluated the immunomodulatory effect of chemotherapy on spheroid-T cells interactions in response to PD-L1 inhibition in triple-negative breast cancer. MCF7 and MDA231 cell lines with differential PD-L1 status were formed into spheroids and used as a tumor model. T cells were activated from PBMCs using ImmunoCult CD3/CD28 T cell Activator. Co-culture assays were performed over 72 hr in a Pu·MA System. The Pu·MA System is an automated microfluidic platform that enables phenotypic and functional assays using physiologically relevant 3D cell models. Cancer spheroids and T cells were cultured, manipulated, and measured in a single well of a microfluidic flowchip. The platform integrates 1) 3D cell model with delivery of immune cells, 2) drug delivery, 3) imaging of phenotypic features and 4) functional profiling for cytokine secretion and viability. T cells-spheroid interaction, invasion and cell viability was assessed using high-content fluorescence imaging CellVoyager CQ1 system. IL2, IFN-γ and TNF-α secretion was measured in collected supernatants using Lumit immunoassays. After viability determination, co-cultures were fixed and stained for cancer and T cell markers (E-Cadherin, F-Actin, CD3, CD8) using automated IF staining protocol and then imaged. Image analysis revealed colocalization and infiltration of the T cells into the spheroids. Disintegration of spheroid, loss of circular shape, and increased number of dead cancer cells indicated T cell mediated cancer cell death. To evaluate the immunomodulatory effect of chemotherapy on spheroid-T cells interactions in response to PD-L1 inhibition, co-cultures were exposed to PD-L1 inhibitor Atezolizumab in the presence or absence of Cisplatin/Pemetrexed. We have analyzed a shift in T cell-mediated killing activity and function in the presence or absence of chemotherapeutics. The proposed co-culture platform can be further extended to a more complex patient-derived 3D models using different cell types. Our functional immune-oncology 3D platform allows to study the crosstalk between immune, cancer and other cell interactions, evaluate new drug candidates and assess individual therapeutic approaches to advance precision medicine. Citation Format: Ekaterina Moroz Nikolov, Anthony Thai, Lila Cooper, Mahomi Suzuki, Arvonn Tully, Rashmi Rajendra, Evan F. Cromwell. Evaluation of the chemotherapy potential to improve the anti-cancer efficacy of PD-L1 inhibition using novel 3D microfluidic co-culture platform. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4559.

DMP1 POSITIVE ASTROCYTES MAINTAIN BLOOD-BRAIN BARRIER VIA TRANSFERRING MITOCHONDRIA TO VASCULAR ENDOTHELIAL CELLS

Though dentin matrix protein 1 (Dmp1) is known to play critical role in mediating bone mineralization, it has also been validated to be expressed in brain and helps maintain blood brain barrier (BBB). Our study aims to clarify the expression pattern of Dmp1 in mouse brain and explore whether intercellular mitochondrial transfer occurs between Dmp1 positive astrocytes (DPAs) and endothelial cells, and thus acting as a mechanism in maintaining BBB during aging.Single cell RNA sequencing (scRNAseq) of 1 month, 6 month, and 20 month old mice brain (n=1, respectively) was employed to identify Dmp1 positive cell types. Dmp1Cre-mGmT and Dmp1Cre-COX8a fluorescent mice were generated to visualize DPAs and investigate their mitochondrial activities. A 3D noncontact coculture system and mitochondrial transplantation were applied to study the role of mitochondrial transfer between astrocytes and bEnd.3 endothelial cells. Dmp1Cre-Mfn2f/f mice were generated by depleting the ER-mitochondria tethering protein Mfn2 in DPAs.Dmp1 was mainly expressed in astrocytes at different ages. GO analysis revealed that cell projection and adhesion of DPAs were upregulated. Confocal imaging on Dmp1Cre-mGmT mice indicated that DPAs are a cluster of astrocytes that closely adhere to blood vessels (n=3). Bioinformatics analysis revealed that mitochondrial activity of DPAs were compromised during aging. Enriched scRNAseq of fluorescent cells from Dmp1Cre-COX8a mice (n=2) and immunofluorescent imaging (n=3) validated the acquisition of extrinsic mitochondria in endothelial cells. 3D coculture of astrocytes and bEnd.3 and direct mitochondrial transplantation revealed the rescue effect of mitochondrial transfer on damaged bEnd.3. BBB was impaired after depleting Mfn2 in DPAs, expressing a similar phenotype with aging brain.Astrocytes that express Dmp1 play a significant role in maintaining BBB via transferring mitochondria to vascular endothelial cells. Compromised mitochondrial transfer between DPAs and endothelial cells might be the potential mechanism of impaired BBB during aging.

Abstract 1807: CB307: a novel targeted CD137 agonist for enhancement of immune cell responses to PSMA+ tumors

Abstract Introduction: CD137 (HGNC:TNFRSF9, 4-1BB) is a TNF receptor superfamily costimulatory receptor expressed by T cells and NK cells. Upon engagement, CD137 enhances immune cell survival, proliferation, cytokine production and memory formation. CD137 agonists in the clinic have shown tantalizing anti-tumor effects, however, hepatic toxicity has hampered clinical utility. To circumvent this limitation, we developed CB307, a half-life extended heavy-chain only Humabody® that selectively agonizes CD137 only in the presence of prostate specific membrane antigen (PSMA, HGNC:FOLH1). PSMA is highly upregulated on tumor cells in prostate cancer and tumor neovasculature in other solid tumors including lung (NSCLC), kidney (clear cell RCC), bladder and colorectal cancers. CB307 enhances immune cell activation in a PSMA-dependent manner. We present here preclinical mechanistic differentiators of CB307 with respect to non-targeted CD137 agonists. Methods: CD137 agonism, immune cell activation and cytokine secretion were evaluated in vitro using coculture assays in the presence/absence of PSMA-expressing tumor cells and in vivo with a transgenic mouse syngeneic tumor model. The ability of CB307 or non-targeted CD137 agonists to enhance tumor cell killing was additionally characterized in 3D spheroid cocultures. In vitro cocultures were used to determine the impact of PSMA expression level on CB307 activity. CB307 was evaluated as a single agent and in combination with PD-1 or PD-L1 inhibitors; tumoral checkpoint contribution was investigated using isogenic PD-L1 positive vs. PD-L1 CRISPR-deleted tumor cells. Results: CB307 or PD-1/PD-L1 inhibition alone induced IL-2 secretion in PBMC/PSMA-expressing tumor cell cocultures; this effect was synergistically enhanced when CB307 was combined with either PD-1 or PD-L1 inhibition. CB307 augmented tumor cell killing in 3D spheroids containing PSMA-expressing cells. Enhanced tumor cell killing was observed in spheroid cultures containing CB307 in combination with PD-1/PD-L1 inhibition. In in vivo pharmacology studies in a syngeneic mouse model, CB307 demonstrated statistically significant inhibition of tumor growth versus control without peripheral T cell activation - consistent with pharmacology restricted to the PSMA+ tumor microenvironment, whereas an anti-CD137 monoclonal antibody induced tumor growth inhibition accompanied by peripheral immune cell proliferation and cytokine production. Summary: CB307 is a novel CD137 agonist that selectively enhances immune cell activity only in the presence of PSMA-positive cells. Our data demonstrate PSMA-targeted CB307 immunological pharmacology both in vitro and in vivo which has supported the ongoing Phase 1 ‘POTENTIA’ clinical trial (NCT04839991). Citation Format: Andrew J. Pierce, Phillip M. Brailey, Sophie Archer, Clare Song, Anca-Giuilia Cionca, Joanne Fernando, Phillip D. Bartlett, Philip Bland-Ward. CB307: a novel targeted CD137 agonist for enhancement of immune cell responses to PSMA+ tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1807.

Abstract 579: Mini-tumors: Pre-clinical HCI assays to screen anti-cancer drugs in the context of organoids, cancer-associated fibroblasts and immune cells

Abstract Introduction Effective drug development relies on representative pre-clinical models to discover and characterize novel cancer therapies. Recapitulating the human specific tumor micro-environment (TME) is essential for IO studies or TME-targeting agents and detection of human-specific (off-)target effects to better predict therapeutic response and clinical outcomes. The recently developed patient-derived organoid (PDO) technology, provides access to representative organotypic cultures, established from patient cancer stem cells. These cultures can be further improved by including additional cell types with strong clinical implications, especially cancer-associated fibroblasts (CAFs) and immune cells. Both CAFs and immune cells from various lineages modulate tumor growth and metastasis, drug resistance and immune-mediated cancer cell killing in the TME and have become popular targets for therapy in the recent decades. Here, we present our improved pre-clinical platform for drug testing, consisting of organotypic mini-tumors combined with high-content imaging screening (HCI) to discriminate different cell types and assess morphological, cytostatic and cytotoxic effects of drugs simultaneously. Methods 3D mini-tumor co-cultures were established of allogenic CAFs and PDOs from pancreatic, colon and breast cancer patients. The mini-tumors were expanded in a TME stimulating medium cocktail that drives myofibroblast expansion and differentiation through PDGFRβ and TGFβ signaling. Subsequently, drug responses to various standard of care (SoC) treatments were determined. Additionally, allogenic PBMCs were included in mini-tumor cultures to assess mini-tumor infiltration and killing. Results Mini-tumor co-cultures were successfully established and characterized in different stimulating cocktails. We found mini-tumor sensitivity to SoC treatment to be altered compared to PDO mono-cultures or co-cultures without TME stimulating factors. Moreover, we observed changes in PBMC infiltration and cancer cell killing in mini-tumors grown in TME stimulating conditions. Conclusion and discussion Taken together, our newly developed pre-clinical mini-tumor HCI platform provides the unique opportunity to screen and test drugs targeting human cancer cells, CAFs or immune cells in vitro in a pathology and TME-resembling context. This will ultimately result in effective target validation and mode of action analysis, timeline and cost reductions, and most importantly, a more efficient entry into the clinic. Citation Format: Marten Hornsveld, Guusje Odenkirchen, Cyrelle van Dort, Jara Garcia Mateos, Saskia de Man, Gera Goverse, Bram Herpers, Mariusz Madej, Leo Price, Marrit Putker. Mini-tumors: Pre-clinical HCI assays to screen anti-cancer drugs in the context of organoids, cancer-associated fibroblasts and immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 579.