Fibroblasts are thought to be key players in the tumor microenvironment. Means to identify and isolate fibroblasts as well as an understanding of their cancer-specific features are essential to dissect their role in tumor biology. To date, the identification of cancer-associated fibroblasts is widely based on generic markers for activated fibroblasts in combination with their origin in tumor tissue. This study was focused on a deep characterization of the cell surface marker profile of cancer-associated fibroblasts in widely used mouse tumor models and defining aberrant expression profiles by comparing them to their healthy counterparts. We established a generic workflow to isolate healthy and cancer-associated fibroblasts from solid tissues, thereby reducing bias, and background noise introduced by non-target cells. We identified CD87, CD44, CD49b, CD95, and Ly-6C as cancer-associated fibroblast cell surface markers, while CD39 was identified to mark normal fibroblasts from healthy tissues. In addition, we found a functional association of most cancer-related fibroblast markers to proliferation and a systemic upregulation of CD87, and CD49b in tumor-bearing mice, even in non-affected tissues. These novel markers will facilitate the characterization of fibroblasts and shed further light in their functions and implication in cancer progression.
Introduction Metastasis to different organs or tissues may require distinct sets of regulators which may influence the homing and growth of tumour cells to specific secondary sites. Under the hypothesis that the immune microenvironment of the different niches may play an important role in this process, we have categorised metastatic samples from different primary tumours based on their immune profile. Material and methods Gene expression data from metastatic samples with different primary tumour origin (n=342) were downloaded from open repository GEO. Samples were scored using different gene expression profiles and characterised on the basis of their immune and stromal infiltration and activation of immune response pathways (Immunophenoscore, MCPcounter, ESTIMATE; among others). Resultant scores were analysed for statistical differences with ANOVA test. Multivariate analysis was used for clustering the samples based in their immune-features. Results and discussions As expected, significant differences were found between the immune profiles of samples metastasizing in distinct organs. For instance, breast cancer metastasis in lung showed a much higher immunogenic score than breast metastasis in brain (p=5e-4), suggesting a different immune microenvironment modulation. Also in breast, significant differences have been found in cell lineages infiltration, lung metastasis being the ones with the highest T cell component (p=0.002) and liver metastasis the ones with the lowest infiltration of endothelial cells (p=0.005). Moreover, in other cancer types like melanoma, samples showed differences among different metastatic locations. Interestingly, when comparing metastatic samples originating from different primary tumour, a high concordance among secondary tumours in immune scores were found; specifically in brain metastasis. These results suggest that cells needs to share similar molecular profiles to evade the immune surveillance and growth in a specific niche, independently of their origin. Conclusion Metastases from the same primary tumour growing in different organs show differences in their immune profile. However, those samples from different primary origin but growing in the same secondary organ shared a characteristic immune profile. These results suggest that immune system plays a role in determining the organ-specific homing of metastasis. Introduction Syngeneic mouse tumour models are widely used to analyse tumour immunology due to their fully competent immune-repertoire and have paved the way for novel immunotherapy agents in multiple tumour entities. However, the amount and composition of tumour infiltrating leukocytes (TIL) is highly variable. This complicates targeted analysis, in particular for small leukocyte subpopulations that may not be analysed properly or lost in the background noise. When working with large cohort sizes, immune-phenotyping by flow cytometry is time consuming and highly work intensive. We have developed improved workflows combining automated tissue dissociation with novel TIL spe...
Cancer cell lines are used to study tumor biology and as models for testing of novel anti-cancer therapeutics. The vast majority of this work has been done using established cell lines that have been cultured for decades. Upon extensive in vitro propagation, cancer cell lines acquire multiple genetic and epigenetic alterations, lose initial heterogeneity present in the parental tumor, and tend to lack tumor initiating as well as multi-lineage differentiation capacity. Consequently, it has been shown that ‘classical' tumor cell lines only insufficiently resemble the characteristics of a tumor. In order to generate improved models for cancer research, novel cell lines are derived from primary tissues and used at low passages. However, this process is very inefficient for most tumor entities. In addition, most of the media used include largely undefined serum, such as FBS, which has been shown to drive primary tumor cell cultures to a more differentiated state when used over multiple passages. We have developed an advanced, serum-free medium for derivation and expansion of tumor cell lines from pancreatic tumors (Noll et al., 2016). This medium has been further optimized concerning formulation, stability, and usability. It allows for efficient generation of primary cell lines from both, patient and xenotransplanted tumors. Primary cell lines derived with this medium closely resembled essential characteristics of the parental tumor, including expression of subtype-specific markers, cellular heterogeneity, as well as genetic and epigenetic signatures. The derived cell lines have been used for genetic engineering and the developed medium allowed for the subsequent expansion of single-cell clones, a pre-requisite for reliable downstream assays. Importantly, culturing with our medium retained the tumorigenic potential as shown by xenotransplantation in immunodeficient mice. The resulting tumors closely and reliably resembled the initial patient tumor. This has been shown on the histomorphological as well as functional level, for example by conservation of tumor specific resistance mechanisms. Taken together, we have developed a serum-free medium for efficient derivation and expansion of tumor cell lines from primary and xenotransplanted pancreatic tumors, allowing for the establishment of easily accessible in vitro models. Moreover, the cell lines could also be used to generate corresponding xenograft models facilitating the translation of in vitro findings directly into a pre-clinical in vivo setting. Citation Format: David Agorku, Anne Langhammer, Elisa M. Noll, Christian Eisen, Silke Schult, Andreas Bosio, Martin R. Sprick, Andreas Trumpp, Olaf Hardt. Efficient derivation and expansion of tumor cell lines from primary and xenotransplanted pancreatic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1050.
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