3D Co-culture Research Articles

3D Coculture between Cancer Cells and Macrophages: From Conception to Experimentation.

A tumor is a complex cluster with many types of cells in the microenvironment that help it grow. Macrophages, immune cells whose main role is to maintain body homeostasis, represent in the majority of cancers the most important cell population. In this context, they are called tumor-associated macrophages (TAMs) because of their phenotype, which contributes to tumor growth. In order to limit the use of animals, there is a real demand for the creation of in vitro models able to represent more specifically the complexity of the tumor microenvironment (TME) in order to characterize it and evaluate new treatments. However, the two-dimensional (2D) culture, which has been used for a long time, has shown many limitations, especially in terms of tumor representation. The three-dimensional (3D) models, developed over the last 20 years, have made it possible to get closer to what happens in vivo in terms of phenotypic and functional characteristics. Due to their architectural similarity to in vivo tissues, they provide a more physiologically relevant in vitro system. Most recently, it is the development of 3D coculture models in which two or three cell lines are cultured together that has allowed a better representation of TME with cell-cell interactions. Unfortunately, there is no clear direction for the design of these models at this time. In this Review on the coculture between cancer cells and TAMs, an in-depth analysis is performed to answer multiple questions on the conception of these models: Which models to use, and with which material and cancer lineage? Which monocyte or macrophage lines should be added to the coculture? And how can these models be exploited?

AML alters bone marrow stromal cell osteogenic commitment via Notch signaling.

Acute myeloid leukemia (AML) is a highly heterogeneous malignancy caused by various genetic alterations and characterized by the accumulation of immature myeloid blasts in the bone marrow (BM). This abnormal growth of AML cells disrupts normal hematopoiesis and alters the BM microenvironment components, establishing a niche supportive of leukemogenesis. Bone marrow stromal cells (BMSCs) play a pivotal role in giving rise to essential elements of the BM niche, including adipocytes and osteogenic cells. Animal models have shown that the BM microenvironment is significantly remodeled by AML cells, which skew BMSCs toward an ineffective osteogenic differentiation with an accumulation of osteoprogenitors. However, little is known about the mechanisms by which AML cells affect osteogenesis. We studied the effect of AML cells on the osteogenic commitment of normal BMSCs, using a 2D co-culture system. We found that AML cell lines and primary blasts, but not normal hematopoietic CD34+ cells, induced in BMSCs an ineffective osteogenic commitment, with an increase of the early-osteogenic marker tissue non-specific alkaline phosphatase (TNAP) in the absence of the late-osteogenic gene up-regulation. Moreover, the direct interaction of AML cells and BMSCs was indispensable in influencing osteogenic differentiation. Mechanistic studies identified a role for AML-mediated Notch activation in BMSCs contributing to their ineffective osteogenic commitment. Inhibition of Notch using a γ-secretase inhibitor strongly influenced Notch signaling in BMSCs and abrogated the AML-induced TNAP up-regulation. Together, our data support the hypothesis that AML infiltration produces a leukemia-supportive pre-osteoblast-rich niche in the BM, which can be partially ascribed to AML-induced activation of Notch signaling in BMSCs.

Abstract C129: Development of new increased potency Ref-1/APE1 targeted inhibitors that show promise for clinical applications against solid tumors

Abstract AP endonuclease-1/Redox factor-1 (APE1/Ref-1 or Ref-1) is a multifunctional protein that is over-expressed in most aggressive cancers and impacts various cancer cell signaling pathways. Among its primary functions, Ref-1’s redox activity plays a significant role in activating transcription factors (TFs) such as NFkB, HIF1a, and AP-1, among others, which are crucial contributors to the development of tumors and metastatic growth. Therefore, development of potent, selective inhibitors to target Ref-1 redox function is an appealing approach for therapeutic intervention. A first-generation compound, APX3330 successfully completed phase I clinical trial in adults with progressing solid tumors with favorable response rate, pharmacokinetics (PK), and minimal toxicity. These positive results prompted us to develop even more potent analogs of APX3330 to effectively target Ref-1 in solid tumors. In this study, we present structure-activity relationship (SAR) identification and validation of more potent lead compounds that exhibit a similar/better safety profile to APX3330. Pharmacokinetics, mouse and human S9 fraction metabolic stability, in silico ADMET properties, ligand-based WaterLOGSY NMR measurements, TF-driven luciferase activity, and two 3-dimensional (3D) cell killing assays (interstitial Tumor-Microenvironment (TME) on a Chip and 3D spheroid) were used for inhibitor characterization. These efforts have elucidated five compounds with improved PK, target engagement, and increased efficacy for cell killing compared to parent compound. Ligand-based WaterLOGSY NMR measurements demonstrate direct interaction of these inhibitors with Ref-1. Target inhibition was further confirmed following Ref-1 treatment via reporter assay with significant and dose-dependent decreases in TF-driven luciferase activity (3-6-fold compared to APX3330). In two 3D co-culture models, second-generation Ref-1 redox analogs suppressed tumor survival significantly with significantly increased potency (~10-fold) compared to APX3330. APX2014, 44, and 51 also inhibited tumor cell proliferation more dramatically (1.5-3.5-fold) than cancer-associated fibroblasts from the TME. Two compounds, APX2044 and 51 outperformed the other analogs as well as the parent compound as assessed by inhibition of downstream TFs regulated by Ref-1 activity, inhibition of mitochondrial metabolism, greater potency against tumors than CAFs in monolayer and in 3D co-culture experiments. PK studies also demonstrated that efficacious doses are clinically achievable for these new inhibitors. Additionally, these findings have allowed us to develop additional analogs for further refinement. Thus, our preclinical findings identified more potent inhibitors of Ref-1 redox function (5-10-fold more than APX3330) that can be translated to the clinic. This study provides invaluable information regarding new Ref-1 analogs for advancement to in vivo and eventual IND-enabling studies and demonstrates the effectiveness of these compounds in sophisticated in vitro models in cancers that are in need of therapeutic options. Citation Format: Silpa Gampala, Hye-Ran Moon, Randall Wireman, Jacqueline Peil, Sonia Kiran, Andi Masters, Christine Bach, Whitney Smith-Kinnamen, Emma Doud, Ratan Rai, Amber L Mosley, Millie M Georgiadis, Bumsoo Han, Chi Zhang, Chafiq Hamdouchi, James Wikel, Mark R Kelley, Melissa L Fishel. Development of new increased potency Ref-1/APE1 targeted inhibitors that show promise for clinical applications against solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C129.

Abstract LB_C19: Suppression of the antitumoral activity of natural killer cells by cancer-associated fibroblasts in a pancreatic TIME-on-Chip model

Abstract Background: Recently, natural killer (NK) cells emerged as a treatment option for various solid tumors. However, the immunosuppressive tumor immune microenvironment (TIME) can reduce the cytotoxic ability of NK cells in pancreatic ductal adenocarcinoma. Cancer-associated fibroblasts within the tumor stroma can suppress immune surveillance by dysregulating factors involved in the cellular activity of NK cells. Herein, the effect of activated pancreatic stellate cells (aPSCs) on NK cell-mediated anticancer efficacy under three-dimensional (3D) coculture conditions was investigated. Methods: 3D cocultures of PANC-1 tumor spheroids (TSs) with aPSCs and NK-92 cells in a collagen matrix were optimized to identify the occurring cellular interactions and differential cytokine profiles in conditioned media using microchannel chips. PANC-1 TSs and aPSCs were indirectly cocultured, whereas NK-92 cells were allowed to infiltrate the TS channel using convective medium flow. Results: Coculture with aPSCs promoted PANC-1 TSs growth and suppressed the antitumor cytotoxic effects of NK-92 cells. Mutual inhibition of cellular activity without compromising migration ability was observed between aPSCs and NK-92 cells. Moreover, the reduced killing activity of NK-92 cells was found to be related with reduced granzyme B expression in NK cells. Conclusions: Herein, a novel TIME-on-chip model based on the coculture of PANC-1 TSs, aPSCs, and NK-92 cells was described. This model may be useful for studying the detailed mechanisms underlying NK cells dysregulation and for exploring future therapeutic interventions to restore NK cell activity in the tumor microenvironment. Citation Format: Hyun-Ah Kim, Hyunsoo Kim, Min-Kyung Nam, Jong Kook Park, Moo-Yeal Lee, Seok Chung, Kyung-Mi Lee, Hyo-Jeong Kuh. Suppression of the antitumoral activity of natural killer cells by cancer-associated fibroblasts in a pancreatic TIME-on-Chip model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C19.

Higher-intensity ultrasound accelerates fracture healing via mechanosensitive ion channel Piezo1.

Osteoporosis-related fractures are a major public health problem. Mechanobiological stimulation utilizing low-intensity pulsed ultrasound (LIPUS) is the most widely accepted modality for accelerating fracture healing. However, recent evidence has demonstrated the ineffectiveness of LIPUS, and the biophysical mechanisms of ultrasound-induced bone formation also remain elusive. Here, we demonstrate that ultrasound at a higher intensity than LIPUS effectively accelerates fracture healing in a mouse osteoporotic fracture model. Higher-intensity ultrasound promoted chondrogenesis and hypertrophic differentiation of chondrocytes in the fracture callus. Higher-intensity ultrasound also increased osteoblasts and newly formed bone in the callus, resulting in accelerated endochondral ossification during fracture healing. In addition, we found that accelerated fracture healing by ultrasound exposure was attenuated when the mechanosensitive ion channel Piezo1 was inhibited by GsMTx4. Ultrasound-induced new bone formation in the callus was attenuated in fractured mice treated with GsMTx4. Similar results were also confirmed in a 3D osteocyte-osteoblast co-culture system, where osteocytic Piezo1 knockdown attenuated the expression of osteoblastic genes after ultrasound exposure. Together these results demonstrate that higher-intensity ultrasound than clinically used LIPUS can accelerate endochondral ossification after fractures. Furthermore, our results suggest that mechanotransduction via Piezo1 mediates ultrasound-stimulated fracture healing and bone formation.

Abstract PR03: 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 a predominant and heterogeneous 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. Citation Format: Ashley M. Mello, Tenzin Ngodup, Yusoo Lee, Katelyn L. Donahue, Jinju Li, Arvind Rao, Eileen S. Carpenter, Howard C. Crawford, Marina Pasca Di Magliano, Kyoung Eun Lee. Hypoxia promotes inflammatory fibroblast formation in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr PR03.

Research of restricted migration evaluation of MDA-MB-231 cells in 2D and 3D co-culture models.

The restricted migration evaluation is conducive to more complex tumor migration research because of the conformity with in vivo tumors. However, the differences between restricted and unrestricted cell migration and the distinction between different evaluation methods have not been systematically studied, hindering related research. In this study, by constructing the restricted environments on chips, the influence of co-culture conditions on the cancer cell migration capacity was studied. The results showed that the restricted channels can discriminate the influence of weak tumor environmental factors on complex tumor migration behaviors by limiting the free growth instinct of tumor cells. Through the comparison of 2D and 3D restricted migration methods, the extracellular matrix (ECM) restriction was also helpful in distinguishing the influence of the weak tumor environmental factor. However, the 3D ECM can better reflect the tortuosity of the cell migration process and the cooperative behavior among cancer cells. In the anticancer drug evaluation, 3D ECM can more accurately reflect the cytotoxicity of drugs and is more consistent with the drug resistance in the human body. In conclusion, the research will help to distinguish different evaluation methods of cancer cell migration, help researchers select appropriate evaluation models, and promote the research of tumor metastasis.

Reconstruction of tracheal window-shape defect by 3D printed polycaprolatone scaffold coated with Silk Fibroin Methacryloyl.

In this study, we aimed to utilize autologous tracheal epithelia and BMSCs as the seeding cells, utilize PCL coated with SilMA as the hybrid scaffold to carry the cells and KGN, which can selectively stimulate chondrogenic differentiation of BMSCs. This hybrid tracheal substitution was carried out to repair the tracheal partial window-shape defect. Firstly, SilMA with the concentration of 10%, 15% and 20% was prepared, and the experiment of swelling and degradation was performed. With the increase of the concentration, the swelling ratio of SilMA decreased, and the degradation progress slowed down. Upon the result of CCK-8 test and HE staining of 3D co-culture, the SilMA with concentration of 20% was selected. Next, SilMA and the cells attached to SilMA were characterized by SEM. Furthermore, in vitro cytotoxicity test shows that 20% SilMA has good cytocompatibility. The hybrid scaffold was then made by PCL coated with 20% SilMA. The mechanical test shows this hybrid scaffold has better biomechanical properties than native trachea. In vivo tracheal defect repair assays were conducted to evaluate the effect of the hybrid substitution. H&E staining, IHC staining and IF staining showed that this hybrid substitution ensured the viability, proliferation and migration of epithelium. However, it is sad that the results of chondrogenesis were not obvious. This study is expected to provide new strategies for the fields of tracheal replacement therapy needing mechanical properties and epithelization.

Overcoming BCR::ABL1 dependent and independent survival mechanisms in chronic myeloid leukaemia using a multi-kinase targeting approach

BackgroundDespite improved patient outcome using tyrosine kinase inhibitors (TKIs), chronic myeloid leukaemia (CML) patients require life-long treatment due to leukaemic stem cell (LSC) persistence. LSCs reside in the bone marrow (BM) niche, which they modify to their advantage. The BM provides oncogene-independent signals to aid LSC cell survival and quiescence. The bone-morphogenetic pathway (BMP) is one pathway identified to be highly deregulated in CML, with high levels of BMP ligands detected in the BM, accompanied by CML stem and progenitor cells overexpressing BMP type 1 receptors- activin-like kinases (ALKs), especially in TKI resistant patients. Saracatinib (SC), a SRC/ABL1 dual inhibitor, inhibits the growth of CML cells resistant to the TKI imatinib (IM). Recent studies indicate that SC is also a potent ALK inhibitor and BMP antagonist. Here we investigate the efficacy of SC in overcoming CML BCR::ABL1 dependent and independent signals mediated by the BM niche both in 2D and 3D culture.MethodsCML cells (K562 cell line and CML CD34+ primary cells) were treated with single or combination treatments of: IM, SC and the BMP receptors inhibitor dorsomorphin (DOR), with or without BMP4 stimulation in 2D (suspension) and 3D co-culture on HS5 stroma cell line and mesenchymal stem cells in AggreWell and microfluidic devices. Flow cytometry was performed to investigate apoptosis, cell cycle progression and proliferation, alongside colony assays following treatment. Proteins changes were validated by immunoblotting and transcriptional changes by Fluidigm multiplex qPCR.ResultsBy targeting the BMP pathway, using specific inhibitors against ALKs in combination with SRC and ABL TKIs, we show an increase in apoptosis, altered cell cycle regulation, fewer cell divisions, and reduced numbers of CD34+ cells. Impairment of long-term proliferation and differentiation potential after combinatorial treatment also occurred.ConclusionBMP signalling pathway is important for CML cell survival. Targeting SRC, ABL and ALK kinases is more effective than ABL inhibition alone, the combination efficacy importantly being demonstrated in both 2D and 3D cell cultures highlighting the need for combinatorial therapies in contrast to standard of care single agents. Our study provides justification to target multiple kinases in CML to combat LSC persistence.

Qpctl Inhibition, By Targeting the Inflammatory Tumor Microenvironment, Constitutes a Novel Therapeutic Approach for Diffuse Large B-Cell Lymphoma

We have previously described that diffuse large B-cell lymphomas (DLBCL) harboring an inflammatory and immunosuppressive microenvironment (i.e., IN-LME) carry independent poor prognosis while proof-of-principle experiments modifying the composition of the LME were sufficient to induce lymphoma regression in mice models ( Kotlov, Cancer Discovery 2021). However, identification of pharmaceutical approaches that by modifying the LME will induce an anti-lymphoma effect while being suitable for short-term human translation remains challenging. The IN-LME is characterized by the presence of pro-inflammatory and immunosuppressive chemokines and cytokines that frequently undergo post-translational modifications. QPCTL (glutaminyl-peptide cyclotransferase-like) is an intracellular enzyme that mediates a N-terminal pyroglutamylation of CD47 and several chemokines, including CCL2 and CCL7, ultimately resulting in their sustained expression and/or secretion. SC-2882 is a first-in-class specific QPCTL inhibitor that induces secondary proteolytic degradation of the monocyte chemoattractants CCL2 and CCL7 ( da Silva, Nature Immunology 2022) and inactivation of the “do-not-eat-me” signal CD47 ( LogtenbergNature Medicine 2019). In a dataset of 470 DLBCL patients we found that the expression of QPCTL correlates negatively with overall survival and progression free survival (0.019 and <0.001, respectively). To determine whether QPCTL could be a potential target in DLBCL, we investigated its expression along with its targets CCL2, CCL7 and CD47 in additional 216 DLBCL cases categorized by LME. We found that while QPCTL and CD47 are expressed at similar levels in all DLBCL cases regardless their COO class and LME category, CCL2 was highly expressed in DLBCL harboring a IN-LME (p <0.05 vs. other LME categories), that also correlate with higher presence of tumor-associated macrophages (TAMs) and exhausted CD8+PD1 HIGH T cells (p <0.01 and p<0.001, vs. other LME categories, respectively). CCL7 was not expressed at detectable levels in majority of LMEs in DLBCL. In preclinical models, murine and human DLBCL cell lines expressed baseline QPCTL and CD47 while CCL2 and CCL7 expression (qPCR) and secretion (ELISA) increased at higher levels upon culturing these cells for 24 h embedded in splenic extracellular matrix 3D cultures. Exposure of a panel of 3 murine and 5 human DLBCL cells to increasing concentrations of SC-2882 (vs. vehicle) for up to 72 h and up to a 10 micromolar dose had no noticeable decrease in cell viability or proliferation. However, SC-2882 (vs. vehicle) at nanomolar concentrations increased anti-Cd20-dependent lymphoma cell phagocytosis by in vitro differentiated macrophages (from splenic monocytes in mice and THP1 cells in humans) in 3D co-cultures (p < 0.05, for all conditions). To further test the anti-lymphoma effect of SC-2882 we used a murine GCB-DLBCL model by implanting GEMM-derived aggressive Ezh2 mutant (EZH2 Y646equivalent) + BCL2 amplified lymphoma cells in the spleen of immunocompetent C57BL/mice (n=14). After implantation, mice were randomized to receive SC-2882 by oral gavage (n=7) or vehicle (n=14) for 14 days. Tumor burden was followed by luciferase imaging (Caliper), and spleens were analyzed at sacrifice by multiparametric imaging, flow-cytometry, and spatial transcriptomics. Oral SC-2882 (vs. vehicle) induced a significant decrease in tumor growth (p=0.0079, Figure 1) without evidence of systemic toxicity by body weight, blood cell count and chemistry panel. Analysis of the LME in remnant splenic tumors showed in SC-2882 (vs. vehicle) treated mice a significant decrease in lymphoma cells and Ki67+ cells (p<0.001) as well as F480+ TAMs (p<0.01) while there was an increase of CD3+ T cell infiltration (p<0.001). We also found a decrease in Cd4+Foxp3+ Tregs and increase in Cd8+Gzb+ cells in SC-2882 treated mice by flow-cytometry. These changes mirrored the findings in the spatial transcriptomics. Additional analysis of LME cell subpopulations by SC-RNA-seq is ongoing. Overall, these data indicate that QPCTL is a potential target in DLBCL, and that QPCTL inhibition exhibits potent anti-lymphoma effects primarily by targeting the IN-LME.

Interferon-Alpha Impairs Neoplastic Vasculogenesis in a 3D iPSC-Based Model of JAK2V617F+ Myeloproliferative Neoplasms

Overt Myelofibrosis (MF) is an end-stage subtype of BCR-ABL1 negative myeloproliferative neoplasms (MPN), which is characterized by progressive bone marrow (BM) fibrosis resulting in loss of normal hematopoietic support as well as inflammatory and vascular complications. While aberrant neoangiogenesis is known to participate in the pathogenesis of MF, Erba et al. (Am J Pathol, 2017) suggested the occurrence of endothelial-to-mesenchymal transition (EndMT) in BM and spleen of MF patients (pts). Given the relevance of endothelial cells (EC) for MF development and the occurrence of JAK2V617F-mutated EC in MPN pts, targeting of the vascular niche has emerged as a promising therapeutic strategy. To untangle the impact of the JAK2V617F mutation on human vasculogenesis, we leveraged high-purity and clonal patient-specific induced pluripotent stem cell (iPSC)-derived EC (iEC) harboring either the JAK2V617F mutation in hetero- (JAK2V617F HET) or homozygosis (JAK2V617F HOM). JAK2 WT and CRISPR-repaired isogenic JAK2 WT (both referred to as JAK2 WT) were used as controls. In a first set of in vitro experiments, JAK2V617F mutated iEC showed a significantly increased proliferation rate in comparison to JAK2 WT iEC. We next evaluated the effect of selected drugs on iEC viability to assess their vascular toxicity. While treatment with 1 µM each of fedratinib, anagrelide or venetoclax led to a decrease in iEC viability (all **p ≤ .01), no significant alterations were observed with 1 µM ruxolitinib (RUX) or 1000 U/ml interferon-alpha (IFNa). Overall, no differences between the 3 genotypes were observed, pointing towards a genotype-independent mode of action. Given the favorable vascular toxicity profile of IFNa, as shown above, its anti-angiogenic potential was then assessed via endothelial tube formation assays. Pre-incubation with 1000 U/ml IFNa prior to seeding of the cells onto a reconstituted basement membrane matrix led to a significant inhibition of capillary-like structures formation in JAK2 WT and JAK2V617F HET or HOM iEC. One of the limitations of 2D culture assays is the lack of 3D homotypic cellular organization, compromising its translational value. Therefore, we established an adaptive 3D fibrin-based model to recapitulate, in vitro, the critical EC-pericyte crosstalk required for formation and maturation of blood vessels. Recent studies highlighted the capacity of BM mesenchymal stem cells (MSC) to differentiate into pericytes. Therefore, we seeded JAK2 WT and JAK2V617F HETor HOM iEC with or without MSC in fibrin hydrogels. MSC and iEC were visualized using different PKH dyes as fluorescent cell linkers to enable live tracking of vascular tree formation. Although self-assembly of JAK2 WT and JAK2V617F HET or HOM iEC into vessel-like structures was observed in the generated monocultures, the presence of MSC in the cocultures led to a significant increase of the formed network area (Fig. 1), hence recapitulating the critical support of pericytes. While acute IFNa exposure did not alter vascular tree formation (Fig. 1), a separate pre-treatment of iEC and MSC prior to cocultivation, significantly impaired neoplastic vasculogenesis (Fig. 2). Interestingly, interactive microscopy image analysis revealed an increased MSC/iEC interaction in JAK2V617F HOM pre-treated cocultures (*p ≤ .05). Bulk RNA-sequencing of JAK2 WT and JAK2V617F HET or HOM iEC treated for 24h with 1000 U/ml showed a strong response to IFNa. Future analysis may unravel a JAK2V617F specific transcription signature. Moreover, we induced EndMT in our 3D iEC-MSC coculture model by treatment with pro-inflammatory cytokines, reflected by the loss of EC markers (e.g. Claudin 5) and upregulation of mesenchymal markers (e.g. aSMA) by immunofluorescence. We are currently evaluating the capacity of RUX vs IFNa to inhibit EndMT. In summary, we show that IFNa impairs neoplastic vasculogenesis in a robust in vitro 3D coculture system that enables precise control of cellularity and genotype. Our model is a valuable tool to decipher the poorly investigated impact of IFNa on the BM microenvironment. In addition, its translational and scalable properties (e.g. by integrating primary or iPSC-derived hematopoietic cells) will allow us to study the impact of additional drugs on MPN-associated fibrotic transformation of the BM.

CNS Invasion of TCF3::PBX1+ Leukemia Cells Requires Upregulation of AP-1 Signaling As Revealed By Brain Organoid Model

Introduction: Involvement of the central nervous system (CNS) remains a challenge in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Currently, the investigation of CNS leukemia mechanisms relies heavily on 2D cell culture and mouse models. However, given the differences between human and murine CNS in cellular identity and architecture, it becomes crucial to explore alternative models to study CNS leukemia. Moreover, novel targets for diagnosis and treatment of CNS ALL are critically needed. Methods: In this study, we established a pioneering 3D co-culture model that combines human induced pluripotent stem cell (iPSC)-derived cerebral organoids with BCP-ALL cells. We developed new methods to extract data from our invasion assays by enhancing the visualization of 3D organoid images, enabling us to accurately measure the invasion depth of leukemia cells compared to healthy controls within the organoids relative to their surface. To gain further insights on leukemia cells invading the organoids compared to those in the non-invaded fraction, we conducted RNA sequencing and immunofluorescence staining. Subsequently, we validated these results in a BCP-ALL in vivo mouse model and in patients initially diagnosed with CNS-positive BCP-ALL compared to CNS-negative cases within a cohort of 100 BCP-ALL patients. Results: Our experiments demonstrated robust and deep engraftment of TCF3::PBX1+ leukemia cell lines and patient-derived xenograft (PDX) cells into cerebral organoids (within a 14-day of co-culture). In contrast, the engraftment of healthy human CD34+ hematopoietic stem and progenitor cells (HSPCs) was limited ( p < 0.05) as compared to leukemia cells. Utilizing the co-culture model, we successfully validated the targeting of CNS leukemia-relevant pathways, such as CD79a/Igα or CXCR4-SDF1, via genetic knockdown or blocking experiments using inhibitor, respectively, which reduced the invasion of BCP-ALL cells into the organoids. Of note, RNA sequencing and immunofluorescence staining analysis revealed a significant upregulation of members of the AP-1 transcription factor complex, namely FOS, FOSB, and JUN, in the organoid-invading cells. Furthermore, we found an enrichment of AP-1 pathway genes in PDX cells recovered from the CNS compared to spleen blasts of mice transplanted with TCF3::PBX1+ PDX BCP-ALL cells (n = 5), thereby supporting the critical role of AP-1 signaling in CNS disease (Figure 1A). In line with these findings, we observed significantly higher levels of the AP-1 gene JUN in patients initially diagnosed with CNS-positive BCP-ALL compared to CNS-negative cases (mean JUN expression: 633.4 ± 78.51 in CNS-negative vs 1142 ± 296.5 in CNS-positive) within a cohort of 100 BCP-ALL patients (Figure 1B). Summary: In summary, we present ALL co-culture systems with iPSC-derived cerebral organoids as a promising complementary model to investigate CNS involvement in BCP-ALL including therapeutic targeting approaches, and identified the AP-1 pathway as a marker of CNS disease in TCF3::PBX1+ BCP-ALL.

Detecting radio- and chemoresistant cells in 3D cancer co-cultures using chromatin biomarkers

The heterogenous treatment response of tumor cells limits the effectiveness of cancer therapy. While this heterogeneity has been linked to cell-to-cell variability within the complex tumor microenvironment, a quantitative biomarker that identifies and characterizes treatment-resistant cell populations is still missing. Herein, we use chromatin organization as a cost-efficient readout of the cells’ states to identify subpopulations that exhibit distinct responses to radiotherapy. To this end, we developed a 3D co-culture model of cancer spheroids and patient-derived fibroblasts treated with radiotherapy. Using the model we identified treatment-resistant cells that bypassed DNA damage checkpoints and exhibited an aggressive growth phenotype. Importantly, these cells featured more condensed chromatin which primed them for treatment evasion, as inhibiting chromatin condensation and DNA damage repair mechanisms improved the efficacy of not only radio- but also chemotherapy. Collectively, our work shows the potential of using chromatin organization to cost-effectively study the heterogeneous treatment susceptibility of cells and guide therapeutic design.

DDDR-27. CLASS I HDAC INHIBITOR AGAINST TM-MEDIATED NETWORK CONNECTIVITY IN GLIOBLASTOMA

Abstract Tumor microtubes (TMs) are long thin cellular protrusions extended by one glioblastoma cell to connect to another and integrate into a communicating network that contributes to glioblastoma’s high therapy resistance. Understanding the biological functions of tumor cell connectivity and overcoming network resistance has emerged as a new therapeutic opportunity. The combinatorial treatment of an anti-TM compound with a cytotoxic treatment has come to focus after it was shown that non-connected glioblastoma cells are more vulnerable to cytotoxic therapy. Besides the development of therapies against specific proteins in TM function, other drugs have also been repurposed and tested for their anti-TM activity and potential additional benefits with radiotherapy. Among these, we tested histone deacetylase (HDAC) inhibitors regarding their potential to overcome TM mediated connectivity and thereby open vulnerabilities in glioblastoma. To investigate the drugs, we applied our drug testing pipeline in vitro and in vivo. Here, we observed fewer TMs in an in vitro monolayer of glioblastoma monoculture after treatment with a class I HDAC inhibitor as opposed to two other compounds from the HDAC inhibitor group. In a 3D co-culture model with human neuronal organoids, treatment with the most promising class I HDAC inhibitor showed a dose-dependent inhibitory effect on cell proliferation and TMs. We then continued to examine the HDAC inhibitor’s effect in our in vivo two photon microscopy model with radiotherapy as cytotoxic co-treatment. After the implantation and establishment of xenograft glioblastoma under a chronic cranial window in mice, treatment with the HDAC inhibitor was started and a course of radiotherapy was added. After six weeks after treatment start, tumors of the co-treated animals were smaller than in animals that were only irradiated. Further studies will involve the examination of the underlying biological mechanism of the anti-TM effects and continued long-term treatment to further improve overall anti-tumor effectivity.

HIPSC model reveals M1 macrophages cause atrial arrhythmia correlated to electrophysiological remodelling of atrial cardiomyocytes in 2D and 3D engineered heart tissue

Abstract Atrial fibrillation (AF) is the most common sustained arrhythmia with an estimated prevalence of 1.5–2%. Current treatments lack effectiveness and do not prevent recurrence. Inflammation is reported in AF patients, however, a causal connection remains elusive. In this study, we investigated the arrhythmic effects of pro-inflammatory macrophages (M1) on human induced pluripotent stem cell (hiPSC)-derived atrial cardiomyocytes (aCM), to better understand their role in AF. Macrophages are known to play a role in cardiac electrophysiology, but their role in arrhythmia is unclear. Three healthy hiPSC lines were differentiated to aCM and M1. Multi-electrode array recordings of isogenic, 2D cocultures of M1 and aCM showed a significant increase in beat rate irregularity compared to control conditions (P<0.001). Furthermore, spike amplitude of aCM and M1 cocultures was significantly decreased (P<0.001). Arrhythmias occurred only after activation of macrophages in the coculture. Addition of anti-inflammatory glucocorticoids significantly reduced beat irregularity in aCM and M1 cocultures compared to vehicle (1.9-fold decrease for Hydrocortisone, P<0.005) further supporting macrophage mediated inflammation to be the cause of the arrhythmia. RNA sequencing of aCM and M1 cocultures revealed aberrant expression of arrhythmia associated cardiac sodium and atrial potassium ion channels (SCN5A, KCNA5). Normal expression was restored through hydrocortisone addition. In addition, inflammation-induced arrhythmia was observed in 3D engineered heart tissues containing hiPSC derived aCM, M1 and cardiac fibroblasts. Tissues showed a significant increase in beating irregularity (P<0.005) compared to controls and had a significant, median reduction (>30%) of contraction amplitude (P<0.05). Our findings suggest a causal relationship between M1 and the occurrence of atrial arrhythmia. The reduction of arrhythmia using glucocorticoids correlates to clinical observations, that show their use being linked to reduced post operative AF burden. These results strongly support the relevance of the proposed hiPSC coculture model and elucidate a new potential AF mechanism.

The inhibitory effect of Gremlin-2 on adipogenesis suppresses breast cancer cell growth and metastasis

BackgroundGremlin-1 (GREM1) and Gremlin-2 (GREM2) are bone morphogenetic protein antagonists that play important roles in organogenesis, tissue differentiation, and tissue homeostasis. Although GREM1 has been reported to be involved in promoting various cancers, little has been reported about effects of GREM2 on cancer. Recently, it has been reported that GREM2 can inhibit adipogenesis in adipose-derived stromal/stem cells. However, as an inhibitor of adipogenesis, the role of GREM2 in cancer progression is not well understood yet.MethodsPre-adipocyte 3T3-L1 cells overexpressing mock or Grem2 were established using a lentiviral transduction system and differentiated into adipocytes-mock and adipocytes-Grem2, respectively. To investigate the effect of adipocyte-Grem2 on breast cancer cells, we analyzed the proliferative and invasion abilities of spheroids using a 3D co-culture system of breast cancer cells and adipocytes or conditioned medium (CM) of adipocytes. An orthotopic breast cancer mouse model was used to examine the role of adipocytes-Grem2 in breast cancer progression.ResultsGrem2 overexpression suppressed adipogenesis of 3T3-L1 cells. Proliferative and invasion abilities of spheroids formed by co-culturing MTV/TM-011 breast cancer cells and adipocytes-Grem2 were significantly reduced compared to those of spheroids formed by co-culturing MTV/TM-011 cells and adipocytes-mock. Compared to adipocytes-mock, adipocytes-Grem2 showed decreased mRNA expression of several adipokines, notably IL-6. The concentration of IL-6 in the CM of these cells was also decreased. Proliferative and invasive abilities of breast cancer cells reduced by adipocytes-Grem2 were restored by IL-6 treatment. Expression levels of vimentin, slug, and twist1 in breast cancer cells were decreased by treatment with CM of adipocytes-Grem2 but increased by IL-6 treatment. In orthotopic breast cancer mouse model, mice injected with both MTV/TM-011 cells and adipocytes-Grem2 showed smaller primary tumors and lower lung metastasis than controls. However, IL-6 administration increased both the size of primary tumor and the number of metastatic lung lesions, which were reduced by adipocytes-Grem2.ConclusionsOur study suggests that GREM2 overexpression in adipocytes can inhibit adipogenesis, reduce the expression and secretion of several adipokines, including IL-6, and ultimately inhibit breast cancer progression.

SILAC proteomics based on 3D cell spheroids unveils the role of RAC2 in regulating the crosstalk between triple-negative breast cancer cells and tumor-associated macrophages

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and is characterized by a high infiltration of tumor-associated macrophages (TAMs). TAMs contribute significantly to tumor progression by intricately interacting with tumor cells. Deeply investigating the interaction between TNBC cells and TAMs is of great importance for finding potential biomarkers and developing novel therapeutic strategies to further improve the clinical outcomes of TNBC patients. In this study, we confirmed the interplay using both 3D and 2D co-culture models. The stable-isotype labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was conducted on 3D cell spheroids containing TNBC cells and macrophages to identify the potential candidate in regulating the crosstalk between TNBC and TAMs. Ras-related C3 botulinum toxin substrate 2 (RAC2) was identified as a potential molecule for further exploration, given its high expression in TNBC and positive correlation with M2 macrophage infiltration. The suppression of RAC2 inhibited TNBC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro. Meanwhile, knocking down RAC2 in TNBC cells impaired macrophage recruitment and M2 polarization. Mechanistically, RAC2 exerted its roles in TNBC cells and TAMs by regulating the activation of P65 NF-κB and P38 MAPK, while TAMs further elevated RAC2 expression and P65 NF-κB activation by secreting soluble mediators including IL-10. These findings highlight the significance of RAC2 as a crucial molecule in the crosstalk between TNBC and TAMs, suggesting it could be a promising therapeutic target in TNBC.

3D bioprinted tumor-vessel-bone co-culture scaffold for breast cancer bone metastasis modeling and drug testing

Bone metastasis is one of the most common complications of advanced breast cancer (BrCa) with a poor clinical prognosis, due to the lack of effective drugs and therapeutics. Traditional screening systems for anti-cancer drugs are not capable of exactly reflecting the efficacy of drugs in vivo, mainly because they cannot replicate the dynamic metastasis process involving primary tumor, blood circulation and bone microenvironment. To precisely simulate the behavior of native BrCa cells, herein, a biomimetic 3D co-culture metastatic model composed of tumor, hollow vascular, and bone tissues is fabricated by one-step 3D bioprinting method for effective drug testing. Gelatin methacryloyl-based photocrosslinkable bioinks containing breast cancer cells (MDA-MB-231), human umbilical vein endothelial cells (HUVECs) and osteoblasts (h-OB) are applied to highly imitate the native metastatic niches with the formation of blood vessels, vascularized tumors, as well as vascularized bone tissues. The dynamic invasion behavior of BrCa cells from original sites to bone tissues through blood vessels is reproduced, and the interactive effect of cancer, vascular and bone cells are investigated. Moreover, the 3D co-culture metastatic model allows for higher drug resistance of BrCa cells than 2D culture and 3D mono-culture models. These results demonstrate that the 3D bioprinted co-culture metastatic model facilitates in-depth understanding of the basic mechanisms of BrCa migration and provides a physiologically relevant platform for the development of new therapeutic drugs.

Analysis of Cellular Crosstalk and Molecular Signal between Periosteum-Derived Precursor Cells and Peripheral Cells During Bone Healing Process Using a Paper-Based Osteogenesis-On-A-Chip Platform.

Periosteum-derived progenitor cells (PDPCs) are highly promising cell sources that are indispensable in the bone healing process. Adipose-derived stem cells (ADSCs) are physiologically close to periosteum tissue and release multiple growth factors to promote the bone healing process. Co-culturing PDPCs and ADSCs can construct periosteum-bone tissue microenvironments for the study of cellular crosstalk and molecular signal in the bone healing process. In the current work, a paper-based osteogenesis-on-a-chip platform was successfully developed to provide an in vitro three-dimensional coculture model. The platform was a paper substrate sandwiched between PDPC-hydrogel and ADSC-hydrogel suspensions. Cell secretion could be transferred through the paper substrate from one side to another side. Growth factors including BMP2, TGF-β, POSTN, Wnt proteins, PDGFA, and VEGFA were directly analyzed by a paper-based immunoassay. Cellular crosstalk was studied by protein expression on the paper substrate. Moreover, osteogenesis of PDPCs was investigated by examining the mRNA expressions of PDPCs after culture. Neutralizing and competitive assays were conducted to understand the correlation between growth factors secreted from ADSCs and the osteogenesis of PDPCs. In vitro periosteum-bone tissue microenvironment was established by the paper-based osteogenesis-on-a-chip platform. The proposed approach provides a promising assay of cellular crosstalk and molecular signal in 3D coculture microenvironment that may potentially lead to the development of effective bone regeneration therapy.

Tumor removal limits prostate cancer cell dissemination in bone and osteoblasts induce cancer cell dormancy through focal adhesion kinase

BackgroundDisseminated tumor cells (DTCs) can enter a dormant state and cause no symptoms in cancer patients. On the other hand, the dormant DTCs can reactivate and cause metastases progression and lethal relapses. In prostate cancer (PCa), relapse can happen after curative treatments such as primary tumor removal. The impact of surgical removal on PCa dissemination and dormancy remains elusive. Furthermore, as dormant DTCs are asymptomatic, dormancy-induction can be an operational cure for preventing metastases and relapse of PCa patients.MethodsWe used a PCa subcutaneous xenograft model and species-specific PCR to survey the DTCs in various organs at different time points of tumor growth and in response to tumor removal. We developed in vitro 2D and 3D co-culture models to recapitulate the dormant DTCs in the bone microenvironment. Proliferation assays, fluorescent cell cycle reporter, qRT-PCR, and Western Blot were used to characterize the dormancy phenotype. We performed RNA sequencing to determine the dormancy signature of PCa. A drug repurposing algorithm was applied to predict dormancy-inducing drugs and a top candidate was validated for the efficacy and the mechanism of dormancy induction.ResultsWe found DTCs in almost all mouse organs examined, including bones, at week 2 post-tumor cell injections. Surgical removal of the primary tumor reduced the overall DTC abundance, but the DTCs were enriched only in the bones. We found that osteoblasts, but not other cells of the bones, induced PCa cell dormancy. RNA-Seq revealed the suppression of mitochondrial-related biological processes in osteoblast-induced dormant PCa cells. Importantly, the mitochondrial-related biological processes were found up-regulated in both circulating tumor cells and bone metastases from PCa patients’ data. We predicted and validated the dormancy-mimicking effect of PF-562,271 (PF-271), an inhibitor of focal adhesion kinase (FAK) in vitro. Decreased FAK phosphorylation and increased nuclear translocation were found in both co-cultured and PF-271-treated C4-2B cells, suggesting that FAK plays a key role in osteoblast-induced PCa dormancy.ConclusionsOur study provides the first insights into how primary tumor removal enriches PCa cell dissemination in the bones, defines a unique osteoblast-induced PCa dormancy signature, and identifies FAK as a PCa cell dormancy gatekeeper.