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Anti-Cancer Activity Profiling of Chemotherapeutic Agents in 3D Co-Cultures of Pancreatic Tumor Spheroids with Cancer-Associated Fibroblasts and Macrophages.

ABSTRACT: Activated pancreatic stellate cells (aPSCs) and M2 macrophages modulate tumor progression and therapeutic efficacy in pancreatic ductal adenocarcinoma (PDAC) via epithelial-mesenchymal transition (EMT). Here, our aim was to analyze the anti-invasion effects of anti-cancer agents where EMT-inducing cancer-stroma interaction occurs under three-dimensional (3D) culture conditions. We used microfluidic channel chips to co-culture pancreatic tumor spheroids (TSs) with aPSCs and THP-1-derived M2 macrophages (M2 THP-1 cells) embedded in type I collagen. Under stromal cell co-culture conditions, PANC-1 TSs displayed elevated expression of EMT-related proteins and increased invasion and migration. When PANC-1 TSs were exposed to gemcitabine, 5-fluorouracil, oxaliplatin, or paclitaxel, 30-50% cells were found unaffected, with no significant changes in the dose-response profiles under stromal cell co-culture conditions. This indicated intrinsic resistance to these drugs and no further induction of drug resistance by stromal cells. Paclitaxel had a significant anti-invasion effect; in contrast, oxaliplatin did not show such effect despite its specific cytotoxicity in M2 THP-1 cells. Overall, our findings demonstrate that the TS-stroma co-culture model of PDAC is useful for activity profiling of anti-cancer agents against cancer and stromal cells, and analyzing the relationship between anti-stromal activity and anti-invasion effects.

SUBMITTER: Jang SD

PROVIDER: S-EPMC8656537 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Recapitulating the tumor microenvironment (TME) in vitro remains a major hurdle in cancer research. In recent years, there have been significant strides in this area, particularly with the emergence of 3D spheroids as a model system for drug screening and therapeutics development for solid tumors. However, incorporating macrophages into these spheroid cultures poses specific challenges due to the intricate interactions between macrophages and cancer cells. To address this issue, in this study, we established a reproducible healthy multicellular 3D spheroid culture with macrophage infiltrates in order to mimic the TME and modulate the drug's efficacy on cancer cells in the presence of macrophages. A 3D spheroid was established using the human cancer cell line CAL33 and THP1 cell derived M0 macrophages were used as a source of macrophages. Cellular parameters including tumour metabolism, health, and mitochondrial mass were analysed in order to establish ideal conditions. To modulate the interaction of cancer cells with macrophage the ROS, NO, and H2O2 levels, in addition to M1 and M2 macrophage phenotypic markers, were analyzed. To understand the crosstalk between cancer cells and macrophages for ECM degradation, HSP70, HIF1α and cysteine proteases were examined in spheroids using western blotting and qPCR. The spheroids with macrophage infiltrates exhibited key features of solid tumors, including cellular heterogeneity, metabolic changes, nutrient gradients, ROS emission, and the interplay between HIF1α and HSP70 for upregulation of ECM degradading enzymes. Our results demonstrate that tumor cells exhibit a metabolic shift in the presence of macrophages. Additionally, we have observed a shift in the polarity of M0 macrophages towards tumor-associated macrophages (TAMs) in response to cancer cells in spheroids. Results also demonstrate the involvement of macrophages in regulating HIF-1α, HSP70, and ECM degradation cysteine proteases enzymes. This study has significant implications for cancer therapy as it sheds light on the intricate interaction between tumor cells and their surrounding macrophages. Additionally, our 3D spheroid model can aid in drug screening and enhance the predictive accuracy of preclinical studies. The strength of our study lies in the comprehensive characterization of the multicellular 3D spheroid model, which closely mimics the TME.

Project description:Purpose: Both cardiomyocytes and cardiac fibroblasts (CF) play essential roles in cardiac development, function, and remodeling. Properties of 3D co-cultures are incompletely understood. Hence, 3D co-culture of cardiomyocytes and CF was characterized, and selected features compared with single-type and 2D culture conditions. Methods: Human cardiomyocytes derived from induced-pluripotent stem cells (hiPSC-CMs) were obtained from Cellular Dynamics or Ncardia, and primary human cardiac fibroblasts from ScienCell. Cardiac spheroids were investigated using cryosections and whole-mount confocal microscopy, video motion analysis, scanning-, and transmission-electron microscopy (SEM, TEM), action potential recording, and quantitative PCR (qPCR). Results: Spheroids formed in hanging drops or in non-adhesive wells showed spontaneous contractions for at least 1 month with frequent media changes. SEM of mechanically opened spheroids revealed a dense inner structure and no signs of blebbing. TEM of co-culture spheroids at 1 month showed myofibrils, intercalated disc-like structures and mitochondria. Ultrastructural features were comparable to fetal human myocardium. We then assessed immunostained 2D cultures, cryosections of spheroids, and whole-mount preparations by confocal microscopy. CF in co-culture spheroids assumed a small size and shape similar to the situation in ventricular tissue. Spheroids made only of CF and cultured for 3 weeks showed no stress fibers and strongly reduced amounts of alpha smooth muscle actin compared to early spheroids and 2D cultures as shown by confocal microscopy, western blotting, and qPCR. The addition of CF to cardiac spheroids did not lead to arrhythmogenic effects as measured by sharp-electrode electrophysiology. Video motion analysis showed a faster spontaneous contraction rate in co-culture spheroids compared to pure hiPSC-CMs, but similar contraction amplitudes and kinetics. Spontaneous contraction rates were not dependent on spheroid size. Applying increasing pacing frequencies resulted in decreasing contraction amplitudes without positive staircase effect. Gene expression analysis of selected cytoskeleton and myofibrillar proteins showed more tissue-like expression patterns in co-culture spheroids than with cardiomyocytes alone or in 2D culture. Conclusion: We demonstrate that the use of 3D co-culture of hiPSC-CMs and CF is superior over 2D culture conditions for co-culture models and more closely mimicking the native state of the myocardium with relevance to drug development as well as for personalized medicine.

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Anti-Cancer Activity Profiling of Chemotherapeutic Agents in 3D Co-Cultures of Pancreatic Tumor Spheroids with Cancer-Associated Fibroblasts and Macrophages.

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