University of North Carolina

Goal: Identify optimal culture conditions for FLC organoids

Principal Investigator: Jian Liu, PhD

Grant length: One year

Study overview: Scientific investigators often use cultures, dishes containing tumor cells, to test candidate treatments for a disease. The type of cultures that most accurately reflect the properties of cells as if they were inside the body are “organoids”, units with traits representative of the whole tissue. FLC organoids are aggregates of FLC tumor cells in a complex along with support cells (such as endothelia, cells associated with vasculature) that behave as a tiny unit of FLC tumor tissue.

In past research, specific defined conditions have permitted the stable formation and maintenance of FLC organoids, but these cultures grow slowly. This study proposed to evaluate novel conditions that hopefully would provide investigators with conditions promoting the development of multiple FLC models. Any complexes identified as biologically active will be made available to investigators elsewhere who will assess if the conditions elicit expansion of FLCs in other FLC model systems.

In this study, specific goals included:

• Aim 1: Identify and synthesize the chains of heparan sulfates, heparins, and/or chondroitin sulfates that bind by high affinity to growth factors (GF) that can drive the expansion of organoids of normal or malignant stem cells.
• Aim 2: Assess the biological effects of a soluble signal complexed with a glycosaminoglycan (GAG) chain (GF/GAG complex) on FLC organoids derived from a UNC-developed PDX tumor line of FLC. If successful, the GF/GAG complexes that are positive will be provided to other investigators to learn if the effects are replicated for other FLC models.

Results: The study found that ten distict heparan sulfate oligosaccharides elicited particular biological responses. Of note, complexes of paracrine signals (a type of cellular communication in which a cell produces a signal to induce changes in nearby cells) and 3-O sulfated HS-oligosaccharides slowed the growth of FLC organoids, and with Wnt3a, stopped the growth of the organoids for months.

Other reported findings included:

• FLC tumors are genetically related to biliary tree stem cell (BTSC) subpopulations, and not to mature hepatic or pancreatic cells
• Gene expression and cell biological phenomena indicate that FLCs are highly enriched for cancer stem cells.
• FLC tumor cells are very difficult to culture outside of the body because of the production by FLC of enzymes that degrade components of the conditions being used for their survival and expansion
• Regulation of FLC cells is dependent on paracrine signals from mesenchymal cell partners (stem cells that differentiate into all types of connective tissue).

The results of this study were published in Matrix Biology in August 2023. The full text of the article, “Fibrolamellar carcinomas–growth arrested by paracrine signals complexed with synthesized 3-O sulfated heparan sulfate oligosaccharides” can be read here.

Implications: The study provided insight into the conditions required to grow and culture FLC tumor organoids. In addition, if future efforts are used to prepare HS-oligosaccharides resistant to breakdown in the body, paracrine signal—HS-oligosaccharide complexes could potential serve as therapeutic agents for clinical treatments of FLC.

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Goal: Validating the RNA signature, expression and function in FLC

Principal Investigator: Praveen Sethupathy, PhD

Grant length: Two years

Study overview: In past work at UNC, a survey of numerous drugs under consideration for FLC use proved non-effective or minimally effective in limiting the growth of the first FLC tumor model. This indicated that completely new strategies are likely to be required to identify effective future therapies for FLC. Consequently, the team sought to define the unique molecular landscape of FLC by analyzing the Cancer Genome Atlas (TCGA) database, a large and growing repository of diverse kinds of data on tumor and matched normal samples across different tumor types. In that data, they identified five genes (PCSK1, CA12, NOVA1, SLC16A14, and TNRC6C), the DNAJB1- PRKACA fusion transcript, and microRNA-10b as the most compelling tissue biomarkers and candidate drivers of FLC tumor progression.

Building on that initial work, this study was focused on:

1. validating the RNA signature of FLC In an independent set of FLC, HCC, and CCA samples;
2. evaluating the expression and function of these RNAs in a UNC-created FLC model; and
3. identifying candidate therapeutic targets of FLC for future clinical development.

Results: This study made substantive progress on several fronts, including:

• Identification of a unique gene (n=16) and ncRNA (n=4) signature of FLC. The key findings of the study were four-­ fold:
  • The team identified FLC samples within TCGA that were mis-­annotated as HCC. Since FLC is a rare liver cancer, each novel cohort provides additional samples for genomic analyses.
  • They compared the expression profile of FLCs with that of ~25 different tumor types within TCGA, the first such pan-­cancer comparative analysis to be performed in FLC.
  • The study showed that long, intergenic non-­coding RNAs (lincRNAs) could stratify FLCs from other liver cancers. They identified four lincRNAs that are uniquely elevated in FLC tumors compared to other liver and non-­liver cancers.
  • The effort demonstrated that expression of CA12 correlates with tumor metastasis, consistent with previously known functions of CA12. This was validated via immunohistochemistry staining in over twenty patient samples.

• Functional studies of candidate FLC oncogenes in tumor spheroids. DNAJB1-­ PRKACA, as well as each of the 20 genes and lincRNAs that were identified as comprising the FLC signature (see above), represent compelling candidates for functional studies. Of those, the team selected three molecules, DNAJB1-­PRKACA, CA12, LINC00313, to evaluate as regulators of FLC tumor phenotypes using a newly developed and characterized patient-­derived xenograft (PDX) model. The team planned continued work on the design and implementation of functional studies to determine whether LINC00313, CA12, and/or DNAJB1-­PRKACA loss-­of-­function influences cellular apoptosis/survival, proliferation, invasive potential, and/or expression of FLC cancer stem cell markers.

• Characterization of FLC exosome small RNA profiles. It has recently been reported that small RNAs, such as microRNAs, in exosomes secreted by tumor cells may be involved in driving tumor development and/or metastasis. The team isolated exosomes from the media of FLC spheroid culture and were able to detect RNA content for small RNA sequencing and analysis.

These results were published in Nature’s Scientific Reports in March 2017 in an article entitled “Comprehensive analysis of The Cancer Genome Atlas reveals a unique gene and non-coding RNA signature of fibrolamellar carcinoma”. The full text of that article can be read here.

Implications: This effort made substantial progress on their goals to characterize a unique molecular signature of FLC and to begin functional evaluation of potential FLC oncogenes using a recently established PDX model. In follow-on work, they planned to focus on continuing the functional studies using asLNAs, as well as on the characterization of FLC exosomal content, with an emphasis on microRNAs.

Note: Information about the PDX model referred to above was published in Nature Communications in their October 2015 publication. The full text of that paper can be read here.

Goal: Understand the developmental biology of fibrolamellar carcinoma

Principal Investigator: Lola Reid, PhD

Grant length: One year

Study overview: Based on extensive work that Dr. Lola Reid’s Laboratory had already performed on normal liver cells and other forms of liver cancer, the goal of the effort was to attempt to define the development biology stage and possibly the origin of fibrolamellar tumors. The work was done in collaboration with pathologists at Sloan Kettering, Columbia University, and Johns Hopkins. If successful the effort would lead to an understanding of where, in the liver developmental pathways, the tumor arose and possibly provide clues of current drugs approaches that might work against FLC.

Results: This study developed new information regarding the stem cell nature and growth requirements of FLC cells. Specifically, the effort:

• Defined conditions for survival and growth of a FLC tumor in vitro and in vivo. These living tumor cells from a FLC patient were hand delivered to the Reid laboratory and have since been successfully grown in different experimental conditions. These tumor lines were placed into immune deficient mice and early data indicated that transplantable tumors may be growing in the mice. If so, such mice would be very valuable to the liver research and pharmaceutical community since they would provide the first animal model in which potential drugs against FLC could be tested.
• Classified FLC tumors as falling into a category of liver cancers comprised of transformed stem cells (cancer stem cells), along with hepatoblastomas, intrahepatic cholangiocarcinomas, and extrahepatic cholangiocarcinomas.
• Complemented the team’s general understanding of liver biology as a system of stem cell-fed maturational lineages and regulated by systemic signals and epithelial-mesenchymal interactions. These themes are summarized in a review that the team published in Hepatology in January 2011 entitled “Human hepatic stem cell and maturational liver lineage biology” (click here to read publication) .

Implications: If correct, the definition of the developmental biology of fibrolamellar cancer cells could lead to an understanding of where, in the liver developmental pathways, the tumor arose and possibly provide clues of current drugs approaches that might work against FLC.

Subsequent work on the growth and culture of FLC cells led to the development of a the first stable, well characterized patient derived xenograft model of FLC. The seed funding of this grant was recognized in the October 2015 publication in Nature Communications of the article “Model of fibrolamellar hepatocellular carcinomas reveals striking enrichment in cancer stem cells” that described that PDX model.

Note: The resulting PDX model is currently being distributed by FCF free-of-charge to any researcher needing a model system for FLC-related studies.

This study was one of four “Freedom of Pursuit” unrestricted research awards to be distributed under the joint FCF/ICARE program active in 2010/2011.