Serum-free Medium Research Articles

Abstract 4251: Establishment of hormone-dependent endometrial tumoroids in a conditioned-medium free, serum-free medium

Abstract Tumoroids, also known as cancer organoids, are self-assembled three-dimensional cultures of patient-derived tumor cells. Compared to traditional 2D cancer cell lines, tumoroids better maintain key characteristics of the original tumor including the genotype and transcriptome, which lead to better in vitro modeling of clinically relevant functional responses such as drug sensitivity. An under-studied aspect of tumoroid cultures has been their ability to recapitulate dependence on hormone signaling in vitro, with some reports indicating hormone dependence is lost over time in culture. To investigate this, we utilized Gibco™ OncoPro™ Tumoroid Culture Medium and defined tissue-specific supplements, FGF10 and beta-estradiol, that when added to the medium enabled the derivation of endometrial tumoroids from tumor resections. Derivation of tumoroid lines compatible with long-term growth depended on a variety of factors, including tissue quality and time from resection to culture initiation. Each of the 15 samples processed formed tumoroids when initially seeded into culture. 11/15 samples reformed tumoroids through multiple passages (typically 1-2 weeks per passage) over 1-3 months in culture. 7/15 samples met our high criteria for stable tumoroid line growth of >5 passages and reaching >5 cumulative population doublings (from initial number of cells seeded post-resection) in culture. Next-generation sequencing of the established tumoroids and the uncultured tumor material demonstrated a high degree of correlation (pearson’s r>0.9) of genomic mutations in a targeted panel; few differentially expressed genes were detected beyond down-regulation of immune-related genes, presumably because the medium is optimized for outgrowth of cancer epithelial cells, and immune cells do not persist during long-term culture. Endometrial tumoroids were capable of growing embedded in basement membrane extract (BME) or in suspension with dilute BME added and could be cryopreserved and recovered. Endometrial tumoroids retained gene expression levels (both presence and absence) of estrogen receptor and progesterone receptor for up to the 20 passages tested in vitro. A subset of the models was tested and showed increased growth rates in response to increased concentrations of beta-estradiol. Of those tested, 2/3 tumoroid cultures stopped growing within 1-2 weeks after removal of beta-estradiol from the medium, demonstrating dependence on this hormone for growth. Maintenance of hormone receptor expression and dependency should enable in vitro studies to understand the signaling mechanisms and potential ways to modulate them in the context of cancer. Overall, this method provides a toolset for studying endometrial cancer, particularly hormone dependency, and may be more broadly applicable to other gynecological or hormone dependent cancers. Citation Format: Chris Yankaskas, Brittany Balhouse, Colin Paul, Pradip Shahi Thakuri, Shyanne Salen, Mark Kennedy, Matt Dallas, David Kuninger. Establishment of hormone-dependent endometrial tumoroids in a conditioned-medium free, serum-free medium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4251.

Abstract 6781: Maintaining the tumor microenvironment: Serum-free cell culture for an advanced 3D in vitro tissue model

Abstract Cancer research has made significant progress in recent years, especially in the field of immunotherapy that uses immune modulators. A significant portion of these novel therapeutic strategies focuses on manipulating the tumor microenvironment (TME). The TME is a complex structure that comprises various cellular elements, including cancer cells, immune cells, and structural components, such as secreted factors and extracellular matrix proteins. However, replicating the human TME in experimental murine models has proved challenging. Therefore, researchers have started to develop ex vivo cultivation protocols of tumor tissues, particularly precision-cut slices (PCS), that offer a three-dimensional representation of the TME. It is essential to select the best culture conditions to ensure the preservation of the TME that closely resembles the in vivo conditions. To further this research, a serum-free cell culture medium has been developed to propagate primary epithelial tumor cells originating from pancreatic, renal, and ovarian tissues. The optimized media formulation was successfully employed in the derivation and expansion of tumor cell lines, sourced from both primary and xenotransplanted tumors. An additional serum-free cell culture medium has been designed to cultivate primary lung cancer cells. Suitable culturing conditions have been introduced that maintain TME composition ex vivo cultured PCS prepared from fresh non-small cell lung carcinoma (NSCLC) tissue. Lung TumorMACS Medium and a serum-containing medium were compared, and it was found that the cellular composition of PCS cultivated in Lung TumorMACS was comparable to the original composition. At the same time, PCS cultivated in Lung TumorMACS showed a lower expression of stress genes, compared to the serum-containing alternative. Citation Format: Alina Siebenmorgen, Christian Wöhle, Katharina Lamfried, Lilian Martinez Carrera, Bianca Heller, Katharina Lupar, Giovanna Bergamini, Daniel Poeckel, Olaf Hardt, Benjamin Theek. Maintaining the tumor microenvironment: Serum-free cell culture for an advanced 3D in vitro tissue model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6781.

Abstract 5438: Circulating tumor cells exhibit a tumor stemness defense phenotype in Head and Neck cancer subjects undergoing cisplatin based chemotherapy

Abstract Introduction: We have proposed that tumor has its own defense mechanism similar to an organism having its own defense at the population level. We also proposed that the center of this tumor defense is the hypoxic niche of cancer stem cells (CSCs). Our lab has previously characterized the hypoxic niche of CSCs, and demonstrated that these CSCs reprogram to a highly inflammatory, aggressive and embryonic stemness phenotype during oxidative stress (1). These reprogrammed CSCs, the tumor stemness defense (TSD) phenotype is transient, and exert altruistic defense against bacterial invasion in SCC-25 cell line (2). Further, VEGF/VEGFR1 autocrine signaling was found in cisplatin induced TSD phenotype (4) and Myc/Hif2alpha stemness pathway was found in TSD phenotype of a leukemia stem cell model (3). We hypothesize that the platinum-induced stress may also activate TSD phenotype in the dormant CSC fraction of head and neck squamous cell carcinoma (HNSCC), which may mobilize to the distant tissue via circulation as circulating tumor cells (CTCs). Methods: To test the hypothesis, we isolated EpCAM+/ABCG2+ CTCs (blood, n=14) and EpCAM+/ABCG2+ CSCs (tumor tissue, n=6/14) of HNSCC subjects under cisplatin treatment. CTCs were collected from peripheral blood mononuclear cells (PBMNC) by the immunomagnetic sorting of EpCAM+ cells, and then cells were expanded in a defined serum free media that we used to maintain the hypoxic CSCs (1). We evaluated the EpCAM+/ABCG2+ CTCs and EpCAM+/ABCG2+ CSCs for TSD phenotype by performing qPCR gene expression, Boyden chamber assay of invasion, clonogenic assay and serial transplantation assay in NOD/SCID mice. Data was compared with the EpCAM+/ABCG2- cell population. We also developed a pre-clinical model of cisplatin-induced reprogramming of HNSCC derived CSCs to understand the molecular mechanisms. Results: In 8/14 patient derived EpCAM+/ABCG2+ CTCs and EpCAM+/ABCG2+ CSCs showed TSD phenotype associated gene expression, high migration/invasion, self sufficiency (ability to form spheroid when grown in serum free media without growth factors), enhanced secretion of protumorigenic growth factors; VEGF, SDF-1, PIGF, and HMGB1. These CTCs and CSCs have shown tumor formation with significant CSC frequency, when injected to NOD/SCID mice. Finally, using a SCC-25 cell line derived pre-clinical model, we demonstrated that cisplatin therapy reprogram CSCs to TSD phenotype. Conclusion: Our work indicates that CTCs with TSD phenotype may indicate the interaction between cisplatin and CSC niche defense. Using the pre-clinical model, the TSD phenotype based CSC niche defense mechanism may be further explored to develop markers for therapy response, metastasis or recurrence in HNSCC.

Abstract 727: FGFR4 promotes colon cancer progression through CAF activation via the CXCL10-CXCR3 axis

Abstract Cancer-associated fibroblasts (CAFs) contribute to cancer progression via crosstalk with cancer and immune cells in the tumor microenvironment (TME). Despite the significant role of CAF, the understanding of the trigger mechanisms of cancer-mediated CAF activation remains unclear. We previously reported the mechanism by which FGFR4 in colon cancer secretes amphiregulin (AREG), resulting in anti-EGFR chemoresistance. It motivated us to investigate whether FGFR4 overexpressing tumors may change the TME, so we explored the mechanism of CAF activation under the FGFR4 signaling pathway. CAF was isolated from BALB/C mice after injecting 1 × 106 CT-26/EV or FGFR4 cells using microbeads and conditioned media (CM) was prepared by filtering after 48h cancer cell culture with serum-free medium In mice model, CT-26/FGFR4 showed more significant tumor progression and CAF abundance (α-SMA, FAP, PDGFR, and vimentin) than CT-26/EV. Additionally, CT26/FGFR4-derived CAFs showed significantly increased invasiveness and contractility, indicating the highly activated status of CAFs. In order to verify the effect of cancer secretome from FGFR4 in cancer cells, normal fibroblast (NIH/3T3) were co-cultured with either stable cancer cells or CM. As a result, CT-26/FGFR4 cells robustly induced the expression of CAF markers in NIH/3T3 cells, indicating the promotion of CAF differentiation from resting fibroblasts. Also, CT-26/FGFR4-derived CM significantly promoted further activation of CAFs compared to CT-26/EV-derived CM. Taken together, FGFR4 contributes not only to CAF differentiation but also to CAF activation via secretome. Next, we performed RNA seq of CT-26/EV and CT-26/FGFR4 cells and identified the upregulated genes related with the interferon (IFN) signaling. Among these genes, we identified IFNβ and CXCL10 as targetable molecules via the activation of TLR3-TBK-IRF and IFN-STAT-CXCL10 signaling pathways. These results were confirmed by RT-PCR and western blot assay. Secreted CXCL10 induced gene expression of CAF markers in fibroblast and increased CAF functions such as migration, invasion and contractibility, reflecting CAF activation. Blocking antibodies and knocking down of CXCL10 inhibited the cancer secretome-mediated CAF markers. The blockade of CXCR3, which is the CXCL10 cognate receptor, also showed an inhibitory effect on the FGFR4-overexpressing cancer-educated CAF. In human colon cancer specimens, the expression of FGFR4, CXCL10, and CAF markers showed a positive correlation with each other. Finally, dual inhibition of FGFR4 and CXCR3 in tumor mouse model suppressed the tumor growth, accompanied by CAF suppression. Our findings reveal the novel mechanism of FGFR4 leading CAF differentiation/activation in TME via the CXCL10-CXCR3 axis. This suggests that FGFR4/CXCR3 inhibitor could be used as a potential therapeutic strategy to attenuate CAF in stromal dominant tumors. Citation Format: Sang-Hee Cho, Hyunjin Bang, Eun-Gene Sun, Ji-Na Choi, Mi-Ra Park, Hyun-Jeong Shim, Jun-Eul Hwang, Woo-Kyun Bae, Ik Joo Chung. FGFR4 promotes colon cancer progression through CAF activation via the CXCL10-CXCR3 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 727.

Abstract 387: Ionizing radiation induces lipid metabolism-associated vulnerabilities in glioblastoma multiforme

Abstract Introduction: Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults. It is highly resistant to its current standard-of-care regimen, which includes surgical resection followed by adjuvant ionizing radiation and temozolomide. Therapy resistance can be attributed to the abundance of genomic alterations in GBM tumors that evolve over time, which contribute to intra-and inter-tumoral heterogeneities that render targeted therapies as inadequate treatment options. Thus, there is an urgent unmet need for effective therapies targeting primary and recurrent GBM. Here, we investigated the impact of ionizing radiation on the transcriptome of patient derived GBM tumor spheres to identify key alterations and biological pathways involved in therapy resistance. Materials and methods: We acquired four independent human GBM tumor sphere cell lines, which were derived from patients and cultured in serum-free media. Tumor spheres were treated with either a sham radiation or a single dose of 10Gy. Using poly-A enrichment whole transcriptome sequencing, we evaluated transcriptional alterations occurring in GBM tumors spheres at 96h post-radiation. To do so, we performed differential expression analysis and gene set enrichment analysis (GSEA) to identify top differentially expressed genes and significant changes in biological phenomena. Additionally, we evaluated the cell viability response of these GBM tumor spheres to radiation. Results and discussion: PCA analysis of the four human GBM tumor sphere cell lines demonstrates that cell type has a substantial impact on variation among samples compared to non-irradiated and irradiated treatment conditions. Upon analysis of genes in common that are either upregulated or downregulated followed by GSEA, we identified enrichment of genes associated with inflammatory responses and ferroptosis repression (e.g., NUPR1, PTGS1, AOX1), and depletion of genes involved in fatty acid metabolism (e.g., INSIG1, PLA2G3, ACAT2). Furthermore, the GBM tumor sphere cell viability responses allowed us to stratify our models into radiosensitive and radioresistant subgroups. Conclusion: Radiosensitive human GBM tumor spheres exhibit transcriptional alterations in genes linked to inflammation and fatty acid metabolism to a greater extent compared to radioresistant tumor spheres. Our study characterizes the radiation response of patient derived GBM models, which is to be leveraged for combating therapeutic resistance. Citation Format: Arianna Richelle Izawa-Ishiguro, Subhiksha Nandakumar, Nicholas Carbone, Annalisa V. Ferrotta, Raashed Raziuddin, Kristen C. Vogt, Daniel A. Heller, Ingo K. Mellinghoff. Ionizing radiation induces lipid metabolism-associated vulnerabilities in glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 387.

Abstract 2779: The significance of TIMP-1 and hypoxia in cancer stemness: A synergistic role in chemoresistance

Abstract Chemotherapeutic resistance in non-small-cell lung cancer (NSCLC) poses a formidable clinical challenge. Numerous studies have highlighted the critical role of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in tumorigenesis and, increasingly, in the development of chemoresistance. We have studied multiple aspects of the MMP-independent, tumor-promoting function of TIMP1 in human NSCLC. Our recent studies have found a pivotal role for TIMP1 in chemoresistance. In order to gain an enhanced comprehension of the mechanisms underpinning TIMP-1-mediated chemoresistance, we have sought to determine the contribution of CSCs and hypoxia. TIMP-1 plays a role in propagating stemness, although relatively unexplored in the context of CSCs within NSCLC. A second crucial factor in the development of chemoresistance in NSCLC is the interplay between intratumoral hypoxia and CSCs. As the tumor grows, metabolic demand for oxygen increases and hypoxic regions are seen in areas furthest from the blood vessels. We have shown TIMP1 levels to increase under experimentally-induced hypoxia. In the present study, we cultured NSCLC cell lines and their TIMP1 KD clones in an assay with serum-free media and found a significant decrease in spheroid size in TIMP1 KD clones. Postulating a reduction in CSC population in KD clones, we determined the NSCLC CSC markers CD133 and ALDH’s expression in the NT and KD TIMP1 clones. Flow cytometry results showed that CD133pos and ALDHhigh populations were significantly decreased in TIMP1 KD clones relative to NT clones. Moreover, KD clone of TIMP1 showed a decrease in the levels of CSC-specific Transcription factors Oct 4A and Nanog. Taken together, our findings confirm that reduction in TIMP1 expression negatively impacts CSC populations. Furthermore, we determined the mRNA levels of CD44, another CSC marker also expressed in lung cancer is upregulated under hypoxia (1% O2) in both the NT and KD clones of TIMP1. Thus, we postulate that TIMP1 enhances chemoresistance by promoting CSC propagation within a hypoxic tumor microenvironment. Citation Format: Ilamathi Thirusenthilarasan, Wei Xiao, Mumtaz V. Rojiani. The significance of TIMP-1 and hypoxia in cancer stemness: A synergistic role in chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2779.

Abstract 3003: Exon skipping for the tumor suppressor Annexin A7 promotes EGFR signaling in stem-like brain tumor initiating cells through receptor recycling

Abstract Background: Glioblastoma (GBM) is one of the most lethal forms of cancer. The majority of GBMs express aberrant receptor tyrosine kinase (RTK) activity, which upon activation, are canonically endocytosed and degraded. This process is disrupted in GBM to promote receptor recycling resulting in hyperactive RTK signaling. We demonstrated that the alternative splicing of the tumor suppressor Annexin A7 (ANXA7) supports the impaired endosomal trafficking of RTKs to favor recycling in GBM. Exon six of ANXA7 can be alternatively spliced to yield two distinct protein isoforms through its inclusion (I1) or exclusion (I2). Methods: We investigated ANXA7 isoform differences for epidermal growth factor receptor (EGFR) trafficking fates first in U251 malignant glioma followed by stem-like brain tumor initiating cells (BTICs) originating from patient-derived xenografts (PDXs). U251s and primary BTICs (JX14) were transduced with lentivirus to overexpress ANXA7 and GFP for enrichment by flow cytometry. JX14 empty vector (EV; I2 only) and I1 (I2 and I1 expressing) cells were plated onto Geltrex and ligand starved overnight in serum-free media to maintain BTICs. We assessed EGFR protein levels following EGF stimulation in a time dependent manner. Immunofluorescence experiments were conducted to measure EGFR-ANXA7 colocalization (Pearson) with markers of the endocytic pathway, which included the early endosomes (EEA1), recycling endosomes (Rab4/Rab11), and lysosomes (LAMP1). EV and I1 cells were also incubated with siRNA targeting both ANXA7 isoforms to determine the impact of ANXA7 loss on endosomal EGFR trafficking. Results: Interestingly, we found drastic differences in endosomal EGFR trafficking in I1 expressing cells. EGFR colocalized with EEA1 in both EV and I1 cells (R2=0.01336, P>0.6275). In EV cells, EGFR was recycled via fast/slow recycling endosomes demonstrated by Rab4/Rab11 colocalization while I1 did not (Rab11: R2=0.9267, P<0.0001). Immunoblotting revealed a significant reduction in EGFR protein in I1 cells only. Furthermore, I1 cells and not EV preferentially degraded EGFR as seen by LAMP1 colocalization (R2=0.9398, P<0.0001). ANXA7 siRNA in EV cells did not alter EGFR trafficking, which implicates I2 as nonfunctional. On the other hand, ANXA7 KD in I1 cells impaired EGFR sorting via a 40% increase (P<0.0010) in total EGFR protein and a 40% reduction (P<0.0001) in colocalization with EEA1. Loss of I1 also shifted the degradation of EGFR back towards recycling. Conclusion: GBMs express I2, which supports EGFR hyperactivity by favoring receptor recycling. I1 overexpression significantly reduced EGFR signaling by promoting degradation. I1-induced reduction in EGFR signaling suggests a new therapeutic vulnerability for BTICs by potentially reducing tumorigenicity or improving the efficacy of EGFR inhibition. Citation Format: Aran Merati, Sindhu Nair, Rajani Rajbhandari, Nicholas Johnson, Markus Bredel. Exon skipping for the tumor suppressor Annexin A7 promotes EGFR signaling in stem-like brain tumor initiating cells through receptor recycling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3003.

Formation of Mycobacterium abscessus colonies in cellular culture in an in vitro infection model

Mycobacterium abscessus is one of the most important nontuberculous mycobacteria that cause lung diseases. In vitro infection models developed to analyze the immune response are frequently based on the addition of mycobacteria to mononuclear cells or neutrophils from peripheral blood. An important requirement of these assays is that most cells phagocytose mycobacteria, only accomplished by using large multiplicities of infection (1 or more bacteria per cell) which may not adequately reflect the inhalation of a few mycobacteria by the host. We propose modifications that try to mimic some of the conditions in which immune cells deal with mycobacteria. For the preparation of the inoculum mycobacteria are grown in solid media followed by preparation to a single cell suspension. Multiplicities of infection (number of bacteria per cell) are below 0.01. Serum-free cellular media is used to allow the growth of M. abscessus. After several days of incubation Bacterial Colonies in Cellular Culture (BCCC) develop, which are enumerated directly under an inverted microscope. These colonies may represent biofilm formation during chronic infections.•Low multiplicity of infection (below 0.01 bacteria per cell) reflects more realistically conditions encountered by immune cells in the lungs.•The surface of mycobacteria prepared for infection assays that are grown in solid media are less affected than that of mycobacteria grown in liquid media with detergents.•Colony formation in the infected cells may reflect the aggregation and biofilm formation in the lungs during chronic infection.

Study of physical processes occurring in serum-containing and polymer-based serum-free cryoprotective media

Study of physical processes occurring in serum-containing and polymer-based serum-free cryoprotective media

Expression of ATP-Binding Cassette Transporter A1 (ABCA1) in Eyelid Tissues and Meibomian Gland Epithelial Cells.

To validate the adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) expression and distribution in human eyelid tissues and meibomian gland epithelial cells. Meibomian gland tissues from human eyelids were isolated by collagenase A digestion and cultured in defined keratinocyte serum-free medium (DKSFM). Infrared imaging was used to analyze the general morphology of meibomian glands. Hematoxylin and eosin (H&E) staining and Oil Red O staining were used to observe the morphological structure and lipid secretion in the human meibomian gland tissues. Quantitative real-time polymerase chain reaction, western blotting, and immunostaining were used to detect the mRNA and protein expression and cytolocalization of ABCA1 in the meibomian gland tissues and cultured cells. The degree of loss of human meibomian gland tissue was related to age. Meibomian gland lipid metabolism was also associated with age. Additionally, human meibomian gland tissues express ABCA1 mRNA and protein; glandular epithelial cells express more ABCA1 mRNA and protein than acinar cells, and their expression in acinar cells decreases with differentiation. Furthermore, the expression of ABCA1 was downregulated in abnormal meibomian gland tissues. ABCA1 was mainly localized on the cell membrane in primary human meibomian gland epithelial cells (pHMGECs), whereas it was localized in the cytoplasm of immortalized human meibomian gland epithelial cells (iHMGECs). The mRNA and protein levels of ABCA1 in pHMGECs were higher than those in iHMGECs. Meibomian gland tissues of the human eyelid degenerate with age. ABCA1 expression in acinar cells decreases after differentiation and plays an important role in meibomian gland metabolism.

Integrated Omics Approach: Revealing the Mechanism of Auxenochlorella pyrenoidosa Protein Extract Replacing Fetal Bovine Serum for Fish Muscle Cell Culture.

The process of producing cell-cultured meat involves utilizing a significant amount of culture medium, including fetal bovine serum (FBS), which represents a considerable portion of production expense while also raising environmental and safety concerns. This study demonstrated that supplementation with Auxenochlorella pyrenoidosa protein extract (APE) under low-serum conditions substantially increased Carassius auratus muscle (CAM) cell proliferation and heightened the expression of Myf5 compared to the absence of APE. An integrated intracellular metabolomics and proteomics analysis revealed a total of 13 and 67 differentially expressed metabolites and proteins, respectively, after supplementation with APE in the medium containing 5%FBS, modulating specific metabolism and signaling pathways, which explained the application of APE for passage cell culture under low-serum conditions. Further analysis revealed that the bioactive factors in the APE were protein components. Moreover, CAM cells cultured in reconstructed serum-free media containing APE, l-ascorbic acid, insulin, transferrin, selenium, and ethanolamine exhibited significantly accelerated growth in a scale-up culture. These findings suggest a promising alternative to FBS for fish muscle cell culture that can help reduce production costs and environmental impact in the production of cultured meat.

Biochemical and functional characterization of heat-inactivated coelomic fluid from earthworms as a potential alternative for fetal bovine serum in animal cell culture

Fetal bovine serum (FBS) plays a pivotal role in animal cell culture. Due to ethical and scientific issues, searching for an alternative, comprising the three R’s (Refinement, Reduction and Replacement) gained global attention. In this context, we have identified the heat inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a potential alternative for FBS. Briefly, we formulated HI-CF (f-HICF) containing serum free medium which can aid the growth, attachment, and proliferation of adherent cells, similar to FBS. In this study, we investigated the biochemical characterization, sterility, stability, formulation, and functional analysis of HI-CF as a supplement in culturing animal cells. Notably, vitamins, micronutrients, proteins, lipids, and trace elements are identified and compared with FBS for effective normalization of the serum free media. HI-CF is tested to be devoid of endotoxin and mycoplasma contamination thus can qualify the cell culture grade. The f-HICF serum free media was prepared, optimised, and tested with A549, HeLa, 3T3, Vero and C2C12 cell lines. Our results conclude that f-HICF is a potential alternative to FBS, in accordance with ethical concern; compliance with 3R's; lack of unintended antibody interactions; presence of macro and micronutrients; simple extraction; cost-effectiveness and availability.

Abstract A114: Robust establishment and expansion of multilineage human fallopian tube organoids in serum-free medium

Abstract High-grade serous ovarian cancer (HGSOC) is the most prevalent subtype of epithelial ovarian cancer. Despite its name, HGSOC originates in the fallopian tube (FT), but metastasizes quickly in the adnexal region and is the most lethal gynecological cancer worldwide. Organoid culture is emerging as a powerful model for studying normal FT cell biology and ovarian cancer. To standardize primary human FT and HGSOC organoid culture, we have developed GyneCult Fallopian Tube Organoid Medium (FTOM), an optimized serum-free medium and a workflow that supports robust and representative FT organoid culture from freshly isolated or cryopreserved primary human FT cells. FT cultures were initiated by seeding 4000 fresh or cryopreserved single cells (8 donors) directly into 20 µL Corning® Matrigel® domes and overlaying them with FT Organoid Medium. Cultures were maintained with full-medium changes every 3 - 4 days and were passaged every 8 - 14 days into new 20 µL Matrigel® droplets, either at a passage ratio of 1:3 or by seeding at 4000 cells per droplet. Cultures were analyzed by immunocytochemistry (ICC) to detect secretory markers keratin 7 (KRT7), oviductal glycoprotein 1 (OVGP1), and PAX8, as well as ciliated cell markers acetylated alpha tubulin (TUBA1A) and FOXJ1. Across all donors, 16 ± 8% (mean ± SD) of dissociated EpCAM+ FT cells formed 50 - 300 µm diameter cystic organoids within 14 days of seeding (n = 8). Cultures can be maintained for at least 5 passages with 14 - 20 cumulative population doublings, at 3 - 4 doublings per passage. ICC analysis confirmed that organoids contained both polarized secretory cells (i.e., KRT7+, OVGP1+, PAX8+) and ciliated cells (i.e. acetylated TUBA1A+, FOXJ1+), indicating multilineage capacity (n = 5). We also tested the compatibility of GyneCult FTOM with culturing HGSOC samples and observed that one of three tumor samples tested generated organoids. These tumour organoids had a solid and irregular shaped morphology and were composed of PAX8+ cells. These cells underwent ≥ 5 cumulative population doublings over 5 passages, with 0.5 - 2 doublings per passage; this performance is consistent with recently published HGSOC organoid culture methods. These results demonstrate that GyneCult Fallopian Tube Organoid Medium is a robust medium for initiating and culturing FT epithelium as organoids, can support HGSOC organoid culture, and is a valuable tool for studying FT biology. Citation Format: Victor Ho, Maggie Chan, Manreet Chehal, Alice Liang, Allen C Eaves, Sharon A Louis, John Stingl. Robust establishment and expansion of multilineage human fallopian tube organoids in serum-free medium [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A114.

Lipolysis inhibition improves the survival of fat grafts through ameliorating lipotoxicity and inflammation.

Fat grafting is a promising technique for correcting soft tissue abnormalities, but oil cyst formation and graft fibrosis frequently impede the therapeutic benefit of fat grafting. The lipolysis of released oil droplets after grafting may make the inflammation and fibrosis in the grafts worse; therefore, by regulating adipose triglyceride lipase (ATGL) via Atglistatin (ATG) and Forskolin (FSK), we investigated the impact of lipolysis on fat grafts in this study. After being removed from the mice and chopped into small pieces, the subcutaneous fat from wild-type C57BL/6J mice was placed in three different solutions for two hours: serum-free cell culture medium, culture medium+FSK (50 μM), and culture medium+ATG (100 μM). Following centrifugation to remove water and free oil droplets, 0.3 mL of the fat particles per mouse was subcutaneously injected into the back of mice. Additionally, the subcutaneous fat grafting area was immediately injected with PBS (control group), ATG (30 mg/kg), and FSK (15 mg/kg) following fat transplantation. Detailed cellular events after grafting were investigated by histological staining, real-time polymerase chain reaction, immunohistochemistry/immunofluorescent staining, and quantification. Two weeks after grafting, grafts treated with ATG showed lower expression of ATGL and decreased mRNA levels of TNFα and IL-6. In contrast, grafts treated with ATG showed elevated expression levels of IL-4 and IL-13 compared to the control grafts. In addition, fewer apoptotic cells and oil cysts were observed in ATG grafts. Meanwhile, a higher CD206+/CD68+ ratio of macrophages and more CD31+ vascular endothelial cells existed in the 2-month ATG grafts. In comparison to the control, ATG treatment improved the volume retention of grafts, and decreased graft fibrosis and oil cyst formation. By preventing oil droplet lipolysis, pharmacological suppression of ATGL shielded adipocytes from lipotoxicity following grafting. Additionally, ATG ameliorated the apoptosis and inflammation brought on by adipocyte death and oil droplet lipolysis in grafted fat. These all indicate that lipolysis inhibition improved transplanted fat survival and decreased the development of oil cysts and graft fibrosis, offering a potential postoperative pharmacological intervention for bettering fat grafting.

The effects of miR-217 inhibitor and mimic in the progression of Kirsten rat sarcoma viral oncogene homologue driven cancers

BackgroundKirsten rat sarcoma viral oncogene homologue (KRAS) is one of the most frequently mutated proto-oncogenes in approximately 90% of pancreatic ductal carcinoma (PDAC) and 45% of colorectal cancer (CC) cases. Studies in the past have identified microRNA-217 (miR-217) as a potential tumour-suppressing miRNA that is downregulated in various cancers. Using in silico prediction algorithms, several studies have identified miR-217 as a potential regulator of KRAS, and we investigated its role in PDAC and CC progression. MethodThe study was carried out in KRAS-driven cancer (KDC) cell lines PANC-1 (pancreatic cancer) and SW-480 (CC), which have mutant KRAS gene expression. The KDC cells are transfected with specific oligonucleotides for miR-217, anti-miR-217, and a negative control in serum-free media using lipofectamine. After fixing the IC50, using specific primers, gene expression studies were carried out by qPCR for KRAS downstream targets and genes associated with apoptosis and cell cycle. Anti-migration and anti-apoptotic effects were studied using the transwell migration assay and annexin-V/PI staining methods, and mitochondrial morphology was observed using a transmission electron microscope. ResultsThe present study demonstrates that overexpression of miR-217 in KDC cells mitigates proliferation and migration and promotes cell cycle arrest and apoptosis of KDC cells via the MAPK/ERK signalling pathway. Besides, decreased miR-217 expression rescues KDC cells from the effects mediated by KRAS downstream signalling. ConclusionThe outcome of the study indicates miR-217 suppresses tumour growth and promotes apoptosis in KDC and that these effects are associated with down regulation of MAPK/ERK signalling.

Current Research, Industrialization Status, and Future Perspective of Cultured Meat.

Expectations for the industrialization of cultured meat are growing due to the increasing support from various sectors, such as the food industry, animal welfare organizations, and consumers, particularly vegetarians, but the progress of industrialization is slower than initially reported. This review analyzes the main issues concerning the industrialization of cultured meat, examines research and media reports on the development of cultured meat to date, and presents the current technology, industrialization level, and prospects for cultured meat. Currently, over 30 countries have companies industrializing cultured meat, and around 200 companies that are developing or industrializing cultured meat have been surveyed globally. By country, the United States has over 50 companies, accounting for more than 20% of the total. Acquiring animal cells, developing cell lines, improving cell proliferation, improving the efficiency of cell differentiation and muscle production, or developing cell culture media, including serum-free media, are the major research themes related to the development of cultured meat. In contrast, the development of devices, such as bioreactors, which are crucial in enabling large-scale production, is relatively understudied, and few of the many companies invested in the development of cultured meat have presented products for sale other than prototypes. In addition, because most information on key technologies is not publicly available, it is not possible to determine the level of technology in the companies, and it is surmised that the technology of cultured meat-related startups is not high. Therefore, further research and development are needed to promote the full-scale industrialization of cultured meat.

Metadherin promotes stem cell phenotypes and correlated with immune infiltration in hepatocellular carcinoma.

Metadherin (MTDH) is a key oncogene in most cancer types, including hepatocellular carcinoma (HCC). Notably, MTDH does not affect the stemness pheno-type or immune infiltration of HCC. To explore the role of MTDH on stemness and immune infiltration in HCC. MTDH expression in HCC tissues was detected using TCGA and GEO databases. Immunohistochemistry was used to analyze the tissue samples. MTDH was stably knocked down or overexpressed by lentiviral transfection in the two HCC cell lines. The invasion and migration abilities of HCC cells were evaluated using Matrigel invasion and wound healing assays. Next, we obtained liver cancer stem cells from the spheroids by culturing them in a serum-free medium. Gene expression was determined by western blotting and quantitative reverse transcri-ption PCR. Flow cytometry, immunofluorescence, and tumor sphere formation assays were used to characterize stem-like cells. The effects of MTDH inhibition on tumor growth were evaluated in vivo. The correlation of MTDH with immune cells, immunomodulators, and chemokines was analyzed using ssGSEA and TISIDB databases. HCC tissues expressed higher levels of MTDH than normal liver tissues. High MTDH expression was associated with a poor prognosis. HCC cells overexpressing MTDH exhibited stronger invasion and migration abilities, exhibited a stem cell-like phenotype, and formed spheres; however, MTDH inhibition attenuated these effects. MTDH inhibition suppressed HCC progression and CD133 expression in vivo. MTDH was positively correlated with immature dendritic, T helper 2 cells, central memory CD8+ T, memory B, activated dendritic, natural killer (NK) T, NK, activated CD4+ T, and central memory CD4+ T cells. MTDH was negatively correlated with activated CD8+ T cells, eosinophils, activated B cells, monocytes, macrophages, and mast cells. A positive correlation was observed between the MTDH level and CXCL2 expression, whereas a negative correlation was observed between the MTDH level and CX3CL1 and CXCL12 expression. High levels of MTDH expression in patients with HCC are associated with poor prognosis, promoting tumor stemness, immune infiltration, and HCC progression.

VX-509 attenuates the stemness characteristics of colorectal cancer stem-like cells by regulating the epithelial-mesenchymal transition through Nodal/Smad2/3 signaling.

Colorectal cancer stem cells (CCSCs) are heterogeneous cells that can self-renew and undergo multidirectional differentiation in colorectal cancer (CRC) patients. CCSCs are generally accepted to be important sources of CRC and are responsible for the progression, metastasis, and therapeutic resistance of CRC. Therefore, targeting this specific subpopulation has been recognized as a promising strategy for overcoming CRC. To investigate the effect of VX-509 on CCSCs and elucidate the underlying mechanism. CCSCs were enriched from CRC cell lines by in conditioned serum-free medium. Western blot, Aldefluor, transwell and tumorigenesis assays were performed to verify the phenotypic characteristics of the CCSCs. The anticancer efficacy of VX-509 was assessed in HCT116 CCSCs and HT29 CCSCs by performing cell viability analysis, colony formation, sphere formation, flow cytometry, and western blotting assessments in vitro and tumor growth, immunohistochemistry and immunofluorescence assessments in vivo. Compared with parental cells, sphere cells derived from HCT116 and HT29 cells presented increased expression of stem cell transcription factors and stem cell markers and were more potent at promoting migration and tumorigenesis, demonstrating that the CRC sphere cells displayed CSC features. VX-509 inhibited the tumor malignant biological behavior of CRC-stem-like cells, as indicated by their proliferation, migration and clonality in vitro, and suppressed the tumor of CCSC-derived xenograft tumors in vivo. Besides, VX-509 suppressed the CSC characteristics of CRC-stem-like cells and inhibited the progression of epithelial-mesenchymal transition (EMT) signaling in vitro. Nodal was identified as the regulatory factor of VX-509 on CRC stem-like cells through analyses of differentially expressed genes and CSC-related database information. VX-509 markedly downregulated the expression of Nodal and its downstream phosphorylated Smad2/3 to inhibit EMT progression. Moreover, VX-509 reversed the dedifferentiation of CCSCs and inhibited the progression of EMT induced by Nodal overexpression. VX-509 prevents the EMT process in CCSCs by inhibiting the transcription and protein expression of Nodal, and inhibits the dedifferentiated self-renewal of CCSCs.

Effects of different concentrations of nicotinamide on hematopoietic stem cells cultured in vitro.

In vitro expansion to increase numbers of hematopoietic stem cells (HSCs) in cord blood could improve clinical efficacy of this vital resource. Nicotinamide (NAM) can promote HSC expansion ex vivo, but its effect on hematopoietic stem and progenitor cells (HSPCs, CD34+CD38) and functional subtypes of HSCs - short-term repopulating HSCs (ST-HSCs, CD34+CD38CD45RACD49f+) and long-term repopulating HSCs (LT-HSCs, CD34+CD38CD45RACD49f+CD90+) is not yet known. As a sirtuin 1 (SIRT1) inhibitor, NAM participates in regulating cell adhesion, polarity, migration, proliferation, and differentiation. However, SIRT1 exhibits dual effects by promoting or inhibiting differentiation in different tissues or cells. We propose that the concentration of NAM may influence proliferation, differentiation, and SIRT1 signaling of HSCs. To evaluate the effects and underlying mechanisms of action of different concentrations of NAM on HSC proliferation and differentiation. CD34+ cells were purified from umbilical cord blood using MacsCD34 beads, and cultured for 10-12 d in a serum-free medium supplemented with cytokines, with different concentrations of NAM added according to experimental requirements. Flow cytometry was used to detect phenotype, cell cycle distribution, and apoptosis of the cultured cells. Real-time polymerase chain reaction was used to detect the transcription levels of target genes encoding stemness-related factors, chemokines, components of hypoxia pathways, and antioxidant enzymes. Dichloro-dihydro-fluorescein diacetate probes were used to evaluate intracellular production of reactive oxygen species (ROS). Determination of the effect of different culture conditions on the balance of cytokine by cytometric bead array. Compared with the control group, the proportion and expansion folds of HSPCs (CD34+CD38) incubated with 5 mmol/L or 10 mmol/L NAM were significantly increased (all P < 0.05). The ST-HSCs ratio and fold expansion of the 5 mmol/L NAM group were significantly higher than those of the control and 10 mmol/L NAM groups (all P < 0.001), whereas the LT-HSCs ratio and fold expansion of the 10 mmol/L NAM group were significantly higher than those of the other two groups (all P < 0.05). When the NAM concentration was > 10 mmol/L, cell viability significantly decreased. In addition, compared with the 5 mmol/L NAM group, the proportion of apoptotic cells in the 10 mmol/L NAM group increased and the proportion of cells in S and G2 phase decreased. Compared with the 5 mmol/L NAM group, the HSCs incubated with 10 mmol/L NAM exhibited significantly inhibited SIRT1 expression, increased intracellular ROS content, and downregulated expression of genes encoding antioxidant enzymes (superoxide dismutase 1, peroxiredoxin 1). Low concentrations (5 mmol/L) of NAM can better regulate the balance between proliferation and differentiation, thereby promoting expansion of HSCs. These findings allow adjustment of NAM concentrations according to expansion needs.

Limonin inhibits the stemness of cancer stem-like cells derived from colorectal carcinoma cells potentially via blocking STAT3 signaling.

Limonin is one of the most abundant active ingredients of Tetradium ruticarpum. It exerts antitumor effects on several kinds of cancer cells. However, whether limonin exerts antitumor effects on colorectal cancer (CRC) cells and cancer stem-like cells (CSCs), a subpopulation responsible for a poor prognosis, is unclear. To evaluate the effects of limonin on CSCs derived from CRC cells. CSCs were collected by culturing CRC cells in serum-free medium. The cytotoxicity of limonin against CSCs and parental cells (PCs) was determined by cholecystokinin octapeptide-8 assay. The effects of limonin on stemness were detected by measuring stemness hallmarks and sphere formation ability. As expected, limonin exerted inhibitory effects on CRC cell behaviors, including cell proliferation, migration, invasion, colony formation and tumor formation in soft agar. A relatively low concentration of limonin decreased the expression stemness hallmarks, including Nanog and β-catenin, the proportion of aldehyde dehydrogenase 1-positive CSCs, and the sphere formation rate, indicating that limonin inhibits stemness without presenting cytotoxicity. Additionally, limonin treatment inhibited invasion and tumor formation in soft agar and in nude mice. Moreover, limonin treatment significantly inhibited the phosphorylation of STAT3 at Y705 but not S727 and did not affect total STAT3 expression. Inhibition of Nanog and β-catenin expression and sphere formation by limonin was obviously reversed by pretreatment with 2 μmol/L colievlin. Taken together, these results indicate that limonin is a promising compound that targets CSCs and could be used to combat CRC recurrence and metastasis.