Chemical, Molecular, and Single-nucleus Analysis Reveal Chondroitin Sulfate Proteoglycan Aberrancy in Fibrolamellar Carcinoma

Fibrolamellar carcinoma (FLC) is an aggressive liver cancer with no effective therapeutic options. The extracellular environment of FLC tumors is poorly characterized and may contribute to cancer growth and/or metastasis. To bridge this knowledge gap, we assessed pathways relevant to proteoglycans, a major component of the extracellular matrix. We first analyzed gene expression data from FLC and nonmalignant liver tissue (n = 27) to identify changes in glycosaminoglycan (GAG) biosynthesis pathways and found that genes associated with production of chondroitin sulfate, but not other GAGs, are significantly increased by 8-fold. We then implemented a novel LC/MS-MS based method to quantify the abundance of different types of GAGs in patient tumors (n = 16) and found that chondroitin sulfate is significantly more abundant in FLC tumors by 6-fold. Upon further analysis of GAG-associated proteins, we found that versican (VCAN) expression is significantly upregulated at the mRNA and protein levels, the latter of which was validated by IHC. Finally, we performed single-cell assay for transposase-accessible chromatin sequencing on FLC tumors (n = 3), which revealed for the first time the different cell types in FLC tumors and also showed that VCAN is likely produced not only from FLC tumor epithelial cells but also activated stellate cells. Our results reveal a pathologic aberrancy in chondroitin (but not heparan) sulfate proteoglycans in FLC and highlight a potential role for activated stellate cells. Significance: This study leverages a multi-disciplinary approach, including state-of-the-art chemical analyses and cutting-edge single-cell genomic technologies, to identify for the first time a marked chondroitin sulfate aberrancy in FLC that could open novel therapeutic avenues in the future.


Introduction
Fibrolamellar carcinoma (FLC) is a rare form of liver cancer that predominantly afflicts adolescents and young adults (1,2). Patients with FLC lack standard of care, leaving surgical resection as the primary therapeutic option. and elevated abundance of glycosaminoglycans (GAG) and associated PGs. GAGs and PGs are of notable interest as they contribute to both the structure and mechanics of the tumor stroma and enhance extracellular signaling by sequestering and concentrating soluble growth factors. Through these mechanisms, GAGs and PGs play an important role in the processes of angiogenesis, proliferation, and migration ultimately promoting tumor metastasis (15)(16)(17)(18).
GAGs are polysaccharides of varying lengths comprised of repeating disaccharide units (19). The most common GAGs are hyaluronic acid (HA), heparan sulfate (HS), and chondroitin sulfate (CS; ref. 3). HA chains bind to many components of the extracellular matrix, including collagen. Unlike HA, HS and CS, which differ in disaccharide composition and glycosidic bond linkage patterns, are conjugated to specific core proteins, which are then reclassified as PGs (20,21). PGs share many general characteristics, including domains that bind soluble growth factors and stimulate cellular receptors. In addition, individual PGs can vary in length, and the core proteins can be alternatively spliced, all of which can have affect physiologic processes (22,23). Finally, polysaccharides can undergo extensive sulfation, the effects of which are essential for these functions (24).
Examples of PGs that have been well studied in the cancer context include HS PG, perlecan (HSPG2), and the CS PG, versican (VCAN). HSPG2 binds multiple FGFs and the VEGF, and is significantly upregulated in many cancers, including melanoma (25,26), breast carcinomas (27,28), and glioblastoma (29). Mouse xenograft models of these cancers in which HSPG2 expression has been ablated show reduced tumor volume (16,28,30). Through similar mechanisms, VCAN is known to promote the metastasis of prostate and breast cancer by promoting platelet-derived growth factor signaling, interacting with selectins, a family of cellular adhesion molecules, and promoting EGFR signaling (31). The role of PGs in liver cancer, particularly hepatocellular carcinoma (HCC), has been studied extensively and multiple HS PGs have been identified as important contributors to tumor progression (32). However, it is unknown whether these mechanisms are shared with FLC.
In this study, we sought to bridge the important knowledge gap on GAGs/PGs in FLC. Specifically, through the combined use of RNA sequencing (RNA-seq), GAG disaccharide quantification by LC/MS-MS, and single-nucleus assay for transposase-accessible chromatin (snATAC), we quantified HS and CS abundance and interrogated the activity of PGs at single-nucleus resolution in FLC and nonmalignant liver (NML) samples. These studies confirmed that FLC tumor cells preferentially produce CS and that VCAN is one of the primary PGs in FLC. Moreover, we demonstrated that there is more ATAC signal at the VCAN locus in activated hepatic stellate cells than any other cell type.

Human Samples
Written informed consent was obtained from all individuals and studies were performed in accordance with ethical guidelines established by the U.S. federal policy for the protection of human subjects (U.S. Common Rule). Tumor and adjacent nonmalignant liver samples were collected from patients with FLC by surgeons in accordance with the Institutional Review Board protocols 1802007780, 1811008421 (Cornell University, Ithaca, NY) and 33970/1 [Fibrolamellar Cancer Foundation (FCF)] and provided by the FCF. Patients included male and female subjects and some samples were collected from the same patient. All samples were deidentified before shipment to Cornell.

PolyA+ RNA Library Preparation and Sequencing
The 27 RNA-seq datasets analyzed in this study were generated previously (5,33). Frozen tumors underwent physical dissociation using a polytron PT1200 E homogenizer (Thomas Scientific) and total RNA was isolated using the Total RNA Purification Kit (Norgen Biotek) per manufacturer's instructions. RNA purity was quantified with the Nanodrop 2000 (Thermo Fisher Scientific) or Nanodrop One and RNA integrity was quantified with the Agilent 4200 Tapestation (Agilent Technologies). Libraries were prepared by the Cornell Transcriptional Regulation and Expression (TREx) Facility using the NEBNext Ultra II Directional RNA kit (New England Biolabs, E7760). Sequencing was performed at the Genomics Facility in the Biotechnology Research Center at Cornell University (Ithaca, NY) on the NextSeq500 (Illumina).

Quantitative PCR
Reverse Transcription was performed using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific). Gene expression was quantified with Taq

Immunoblot Analysis
FLC and NML tissues were lysed in RIPA buffer containing Halt protease and phosphatase inhibitors (Thermo Fisher Scientific) at 4°C. Lysates were incubated for 30 minutes and centrifuged at 14,000 × g for 10 minutes at 4°C. Total protein in the supernatant was quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific). Samples were denatured in NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) containing 5% β-Mercaptoethanol for 10 minutes at 70°C and loaded to a 12% SDS-polyacrylamide gel. After electrophoresis, samples were transferred to polyvinylidene difluoride membrane and blocked in TBS containing 0.5% TWEEN20 (TBST) and 3% BSA for 1 hour at room temperature. Membranes were probed for anti-VCAN GAGβ (1:1,000 dilution, rabbit source, Millipore AB1033) or anti-vinculin (1:10,000 dilution, mouse source VLN01, Thermo Fisher Scientific MA5-11690) overnight at 4°C and then incubated with goat anti-rabbit horseradish peroxidase-linked IgG (1:10,000, Cell Signaling Technology). Membranes were visualized using a ChemiDoc MP (Bio-Rad).

Immunohistofluorescence Analysis
FLC and NML tissues were formalin fixed at the time of surgery, dehydrated in ethanol, and paraffin embedded for tissue sectioning onto glass slides. Tissue was deparaffinized by two incubations in xylene, followed by one incubation in 1:1 xylene:ethanol (3 minutes per incubation). Tissue was rehydrated by incubation in decreasing concentrations of ethanol: twice in 100%, 95%, 75%, and 50% (3 minutes per incubation). Finally, tissue was incubated in a flushing water bath for 15 minutes. Tissue was then incubated in methanol for 20 seconds, followed by equilibration in PBS for at least 2 minutes. Antigen retrieval was performed by incubating for 20 minutes in preheated 10 mmol/L sodium citrate (pH6.0) buffer containing 0.05% tween-20 (weight/volume) submerged in a boiling water bath. Tissue in citrate buffer was then removed for the bath and allowed to cool for 30 minutes. Tissue was then incubated in PBS containing 0.03% tween-20 for 20 minutes followed by blocking in 10% normal goat serum diluted in PBS containing 0.03% tween-20 for 1 hour. Tissue was then washed in PBS containing 0.03% tween-20 for 2 minutes following overnight incubation at 4°C with anti-VCAN GAGβ (1:100 dilution, rabbit source, Millipore AB1033). Tissue was then washed three times in PBS containing 0.03% tween-20 for 15 minutes followed by secondary antibody staining for 1 hour at room temperature (anti-rabbit Alexafluor 488, 1:1,000 dilution, goat source, Thermo Fisher Scientific A32731). Tissue was washed three times in PBS containing 0.03% tween-20 for 15 minutes followed by a 5-minute incubation with 4 ,6-diamidino-2-phenylindole (DAPI) to counterstain cell nuclei. Excess DAPI was washed out with two incubations in PBS for 10 minutes each. Tissue was then dried briefly, a small volume VectaMount (Vector Labs, H-5000) of was added, and a coverslip was mounted. Images were acquired on an Olympus DP80 microscope with the CellSense Dimension software package. All images received equal brightness balancing with ImageJ software.
The homogenate was incubated for 3 minutes on ice and then mixed by pipetting 10 times. A total of 6 mL of ATAC-RSB wash buffer (1× ATAC-RSB: 10 mmol/L TRIS pH 7.4, 10 mmol/L sodium chloride, 3 mmol/L magnesium chloride) was added and the homogenate was mixed by pipetting five times, incubated on ice for 5 minutes, and centrifuged at 500 × g for 10 minutes at 4°C, and the supernatant was removed without disrupting pellet. The nuclei were resuspended in 2 mL of ATAC-RSB wash buffer and then dissociated with a Dounce homogenizer using a loose pestle for five strokes, and then a tight pestle for 15-20 strokes, and incubated on ice for 3 minutes. Then 6 mL of ATAC-RSB wash buffer was added mixed by pipetting 10 times and incubated on ice for 3-5 minutes. The suspension was passed through 70-μm cell strainer, centrifuged at 500 × g for 10 minutes at 4°C, and the supernatant was removed.

Tn5 Storage and Transposome Assembly
Tn5 transposase (4 μmol/L) is stored at −80°C and diluted for usage by adding 0.8 volume of 100% glycerol. Tn5 transposomes were assembled by adding 0.11 volume of barcoded Tn5 adaptors (25 μmol/L stock solution) to Tn5 stock solution. The mixture was incubated at room temperature for 12-24 hours. The transposome (∼2 μmol/L) can be used directly or stored at −20°C.

Tagmentation and Sample Processing
Combinatorial single-nucleus barcodes were generated using a strategy modified from ref. 34 and developed in the Genomics Innovation Hub at Cornell.
Nuclei suspension (8 μL) was distributed onto 96-well plates and 1 μL of each i5 and i7 transposome (final concentration 400 nmol/L), was added to each well, resulting in 96 combinations of Tn5 barcodesper plate. The tagmentation reaction plate was incubated (30 minutes at 50°C) and the reaction was terminated by adding 10 μL 20 mmol/L EDTA (15 minutes at 37°C). Next, 20 μL of Sorting buffer (1× SB: 1× PBS, 2 mmol/L EDTA, 20 ng/mL BSA) was added to each well and nuclei were repooled into a single sample. Intact nuclei were then stained with DRAQ7 for 15 minutes (ABCam, ab109202), passed through a 30 μm filter, and reisolated by FACS using a FACSMelody instrument (Becton, Dickinson).
A 96-well destination PCR plate was preloaded with 10 μL of modified sorting buffer (1× SEB: 10 mmol/L TRIS pH 8.0, 12 ng/μL BSA, 0.05% v/v SDS), 25 nuclei were distributed into each well, and incubated for 10 minutes at 55°C to disrupt Tn5. We added 2.5 μL of 5% v/v Triton-X100 per well to neutralize the SDS prior to PCR. Libraries were amplified 15 cycles with a custom universal P5 primer and a barcoded P7 primer (1 μL of 25 μmol/L primer in 25 μL PCR reaction per well).

PCR Cleanup, Size Selection, and Sequencing
All wells were repooled and purified with a MinElute PCR purification kit following the manufacturer's instructions (Qiagen, 28004) and eluted twice with 20 μL of the supplied buffer. The 40 μL elution was further purified and size selected using magnetic solid phase reversible immobilization (35) beads following the manufacturer's instructions (Beckman Coulter, A63880) and eluted in 20 μL. Libraries were sequenced on the HiSeq platform (1 lane) at Novogene.

snATAC-seq QC and Dimensionality Reduction and Clustering Analysis
The transcription start site (TSS) enrichment score and fragment number of each nucleus is calculated using ArchR (41) v1.0.1. Nuclei with TSS enrichment score less than 3 and fragment number less than 1,000 are removed. Doublet scores were calculated with default parameters.
We preformed iterative latent semantic indexing by using the "addIterativeLSI" function of ArchR. We then used the default harmony algorithm to correct for batch-effects differences and added clusters using the "addClusters" function.

Identification of Marker Features
We identified cluster markers using the function "getMarkerFeatures" with default parameters and then applied the "addImputeWeights" function to impute

AACRJournals.org
Cancer Res Commun; 2(7) July 2022 the weights of markers. We visualize marker features using the 'plotEmbedding' function. To plot browser tracks, we used the "plotBrowserTrack" function and arranged track rows from highest to lowest accessibility using the "useGroups" parameters.

Identification of Cell Types from snATAC-seq Data
We used unbiased approaches to assign cell-type identity to clusters. A pairwise comparison of ArchR-defined markers and single-cell RNA-seq liver markers revealed high-confidence cell-type assignments for clusters 1-3, and 5-12. Additional Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses confirmed several cluster assignments.

Plotting Browser Tracks
Accessibility of chromatin surrounding genes of interest is plotted using the default "plotBrowserTrack" function.

RNA-seq Bioinformatic Analysis
Paired end RNA-seq reads were aligned to the human genome (hg38) using STAR (v2.4.2a) and reads aligning to the transcriptome were quantified using Salmon (v0.6). Differential expression was determined with DESeq2.0 (v1.3) using a model that accounts for sequencing facility as a covariate.

Statistical Analysis
Statistical comparisons of quantitative PCR and immunoblot results were made using Student t test. Significant differences in gene expression were determined using DESeq2.0. Graphs were generated in the R software package and error bars represent the SE.

Preparation of 13 C-labeled CS Disaccharide Calibrants
Four 13 C-labeled CS disaccharides were prepared from three 13 C-labeled CS 8-mers, including 13 C-labeled CS-A 8-mer, 13 C-labeled CS-C 8-mer, and 13 Clabeled CS-E 8-mer, as described in Supplementary Fig. S1A. The synthesis of 8-mers was completed using the enzymatic approach (42,43). The only exception from previously published procedures is that a 13 C-labeled UDP-GlcA was used to replace unlabeled UDP-GlcA during the synthesis to introduce the 13 C-labeled GlcA residue to the 8-mer products. The synthesis of UDP-[ 13 C]GlcA was completed enzymatically from [ 13 C]glucose as described previously (44,45). The structures of 8-mer products were confirmed by electrospray ionization mass spectrometry. overnight. The extent of reaction completion was monitored by the strong anion exchange chromatography on a Pro Pac PA1 column (9 × 250 mm, Thermo Fisher Scientific) by measuring the absorbance at 232 nm. The purification of 13 C-labeled CS disaccharides was performed on the Q-Sepharose fast flow column. Mobile phase A was 20 mmol/L NaOAc, pH 5.0 and mobile phase B was 20 mmol/L NaOAc and 1 mol/L NaCl, pH 5.0. The elution gradient with a flow rate of 1 mL/minute was used. The absorbance at 232 nm was scanned and recorded. After purification, the disaccharides were desalted on a Sephadex G-10 column. The quantification of 13 C-labeled disaccharide calibrants was performed on the basis of the standard curve of commercially available native CS disaccharide standards (Iduron).

Structure Analysis of 13 C-Labeled Disaccharide Calibrants
A strong anion exchange column Pro Pac PA1 (9 × 250 mm, Thermo Fisher Scientific) was used to determine the purity of 13

Quantification of the 13 C-Labeled Disaccharide Calibrants
The CS native disaccharides (Iduron) were dissolved in water and diluted to the concentration of 5, 10, 20, 40, and 80 μg/mL. A total of 50 μL of the diluted CS was injected into HPLC to make the standard curve to quantify the 13 C-labeled disaccharide calibrants. The stock solutions of four 13 C-labeled disaccharide calibrants were diluted to 10 or 20 times and then 50 μL was injected to the HPLC analysis. The concentration of the 13 C-labeled disaccharide calibrants stocks were determined by comparing the peak areas at 232 nm with unlabeled disaccharides.

Linear Dynamic Range Determination
Individual stock solutions of four CS unlabeled disaccharides (Iduron) were The linear dynamic range working solutions containing 13 C-labeled internal standard were freeze dried and reconstituted in the 20-μL mouse plasma.
The reconstituted solutions were filtered by passing through a YM-3KDa spin column (Millipore) and washed twice with deionized water to recover the disaccharides in the eluent. The AMAC (2-aminoacridone) derivatization of lyophilized disaccharides was carried out by adding 6 μL of 0.1 mol/L AMAC solution in DMSO/glacial acetic acid (17:3, v/v) and incubating at room temperature for 15 minutes. Then 6 μL of 1 mol/L aqueous sodium cyanoborohydride (freshly prepared) was added to this solution, where AMAC represents AMAC purchased from Sigma-Aldrich. The reaction mixture was incubated at 45°C for 2 hours. After incubation, the reaction solution was centrifuged to obtain the supernatant that was subjected to the LC-/MS-MS analysis.

LC/MS-MS Analysis
The analysis of AMAC-labeled disaccharides was performed on a Vanquish Flex UHPLC System (Thermo Fisher Scientific) coupled with TSQ Fortis triple-quadrupole mass spectrometry as the detector. The C18 column (Agilent InfinityLab Poroshell 120 EC-C18 2.7 μmol/L, 4.6 × 50 mm) was used to separate the AMAC-labeled disaccharides. Mobile phase A was 50 mmol/L ammonium acetate in water. Mobile phase B is methanol. The elution gradient of from 5%-45% mobile phase B in 10 minutes, followed by isocratic 100% mobile phase B in 4 minutes and then isocratic 5% mobile phase B in 6 minutes was performed at a flow rate of 0.3 mL/minute. Online triplequadrupole mass spectrometry operating in the multiple reaction monitoring mode was used as the detector. The ESI-MS analysis was operated in the negative-ion mode using the following parameters: negative-ion spray voltage at 4.0 kV, sheath gas at 45 Arb, aux gas 15 arb, ion transfer tube temperature at 320°C and vaporizer temperature at 350°C. TraceFinder software was applied for data processing. The normalized peak areas of the 13 C-labeled calibrants were plotted against the concentrations of linear dynamic working solutions. water. The collected filtrates were freeze dried before the AMAC derivatization. The AMAC label and LC/MS-MS analysis of the collected disaccharides of tissues was performed as described above. The amount of tissue CS was determined by comparing the peak area of native disaccharide to each calibrant. HS was extracted from two NML tissues and four FLC tissues. The method for the analysis of HS followed the procedures described in a previous publication (46). Three CS disaccharides, including Ddi-2S, Ddi-2S6S, and Ddi-2S4S, were only subjected to relative quantitation as the 13 C-labeled disaccharides were unavailable. Standard curves of these three disaccharides were generated using unlabeled disaccharide standards that were purchased from Iduron.

Data Availability
Previously published (33) RNA-seq data can be downloaded from Gene Expression Omnibus (GEO) using the following GEO accession number: GSE181922.
Previously published (5) RNA-seq and chromatin run-on sequencing (ChROseq) can be downloaded from the European Genome-Phenome Archive (EGA) using the following EGA accession number: EGAS00001004169. Single-nucleus assay for transposase-accessible chromatin followed by sequencing (snATACseq) bam and tabix data files generated in this study have been deposited in the GEO and are accessible through the accession number GSE202315.

Schematics
Chemical structures were created with Chemdraw (by PerkinElmer). Schematics were created with BioRender.com.

Chondroitin but not HS Biosynthesis Genes are Increased in FLC
Tissue samples from patients with FLC were acquired at the time of surgical procedures through a collaboration with the FCF and subjected to RNA extraction and messenger RNA-seq (n = 23 tumor samples and n = 4 adjacent NML samples), as reported previously (5). We then performed an analysis of differential gene expression for enzymes related to GAG biosynthesis, focusing on HA, HS, and CS (3).
The expression of HA synthase 1-3 in FLC did not meet our standard threshold of robust expression (>500 normalized counts), and therefore the HA pathway was not considered for further analysis. We then assessed the expression of genes which catalyze the formation of the common tetrasaccharide linker required for HS and CS PG production (Fig. 1A). The initial addition of xylose to serine residues in polypeptide chains is catalyzed by xylosyltransferases (XYLT or XYLT; ref. 47). This reaction is followed first by the addition of two galactose molecules [catalyzed by β1,4-galactosyltransferase-I (BGALT) and β1,3-galactosyltransferase-I (BGALT)], and subsequently by the addition of glucuronic acid [catalyzed by β1,3-glucuronyltransferase-I (BGALT)] (48,49). We found that the expression in FLC of the genes that code for these enzymes meets the threshold criteria but is not significantly different relative to NML.
We next interrogated those genes encoding enzymes responsible for HS and CS polymerization. HS chain formation begins with the addition of Nacetylglucosamine to the common linker by N-acetylglucosaminyltransferase (EXTL), followed by the addition of glucuronic acid (GlcA) by exostosin glycosyltransferases (EXT and EXT) to create the HS disaccharide (ref. 20; Fig. 1A). We found that the expression of EXTL and EXT is unchanged in FLC, and that EXT is significantly decreased (Fig. 1B). In addition, HS chains undergo deacetylation and sulfation, catalyzed by the enzyme Ndeacetylase and N-sulfotransferase 1 (NDST; ref. 50), as a critical maturation step and there is no significant change in the expression of this gene in FLC (Fig. 1B).   P = 7.6 × 10 −9 ) in FLC compared with NML (Fig. 1C). The expression levels of CSGALNACT, CHPF, and CHSY are unchanged in FLC; however, CHPF is more abundant in FLC than any of the HS polymerizing factors (Fig. 1C).

CS chain elongation begins with the addition of N-acetylgalactosamine
Given that HS and CS chains share a common linker, the stoichiometric ratio of EXTL and CSGALNACT enzymes is the primary factor determining whether HS or CS chains will be generated (52). We compared the expression of CS-GALNACT with EXTL within each of the FLC and NML samples and found that CSGALNACT is on average approximately 4.5 times (P = 0.017) more abundant than EXTL (Fig. 1D) in FLC samples, while being roughly equal in NML samples (0.77-fold). In an independent cohort of patients (FLC n = 11 and NML n = 4), we confirmed by qRT-PCR that CSGALNACT expression is increased >10-fold (Fig. 1E). A similar result was obtained when the analysis was restricted only to matched patient samples (Fig. 1F).

CS Chains are Aberrantly Elevated in FLC
The gene expression analysis is strongly suggestive of increased CS, but not HS, abundance in FLC. To test this hypothesis, we quantified HS and CS abundance in FLC using a novel chemical analytic method. Because of the relatively low abundance of CS from biological tissues, a new quantitative CS analytic method with high sensitivity was developed for this study. Disaccharide analysis is a commonly used approach to analyze the structure of CS polysaccharides.
The method involves the degradation of CS polysaccharides into disaccharides using chondroitin ABCase, and the resultant disaccharides were subjected to LC/MS-MS analysis ( Fig. 2A). Furthermore, summing up the amounts of individual disaccharides from the digested CS provides the total amount of CS.
To increase the quantitation capability, we employed four 13 C-labeled CS disaccharide calibrants as internal standards, including di-0S, di-4S, di-6S, and di-4S6S ( Supplementary Fig. S1). The 13 C-labeled CS disaccharide calibrants were obtained from three uniquely designed 13 C-labeled CS octasaccharides (8-mers) that were synthesized by an enzymatic approach. The di-4S disaccharide is found in CS-A polysaccharide, whereas di-6S and di-4S6S are found in CS-C and CS-E polysaccharides, respectively. The di-0S disaccharide is found in all subtype CS polysaccharides from biological sources. The inclusion of 13 C-labeled calibrants eliminated batch-to-batch variations, increasing the data consistency ( Supplementary Fig. S1B-E).
The sulfation of CS chains is mediated by a family of chondroitin sulfotransferase (30)  O sulfotransferase (CHST). We found that although the expression of CHST in FLC is lower than that of CHST, its levels are also significantly increased compared with NML (2.3-fold, P adjusted = 0.02; Fig. 3E). Likewise, although CS di-6S is not as abundant as CS di-4S, it is similarly increased in FLC (9-fold, P = 0.023; Fig. 3F).
( Fig. 3J) is generated by uronyl 2-O-sulfotransferase (UST) activity. We found that the expression of UST is unchanged (Fig. 3K) and the abundance of CS di-2S4S is unchanged (Fig. 3L) in FLC. We performed a similar quantification for HS and found that none of the sulfated subtypes are significantly altered in FLC (Supplementary Fig. S2A-G), consistent with the finding that total HS abundance is unchanged in FLC (Fig. 1D). In an independent patient cohort, we found by qRT-PCR analysis that the expression of CHST is significantly increased in FLC in all samples ( Supplementary Fig. S3A) as well as in matched samples only (Supplementary Fig. S3B) and that CHST is trending upward in FLC ( Supplementary Fig. S3C and D). Taken   chemical findings strongly indicate that FLC tumors are marked by aberrant levels of total CS as well as specific sulfated subtypes.

VCAN is the Primary CS-associated Protein in FLC
We pairs confirmed dramatic elevation of VCAN protein in FLC (average ∼200fold; Fig. 4D and E). Specifically, VCAN protein is variable but abundant in the tumor tissue from all 3 patients with FLC, while virtually absent in the adjacent nonmalignant samples (Fig. 4D, bottom). Finally, we performed immunohistofluorescent (IHF) staining on two matched FLC/NML pairs of samples and confirmed that VCAN protein is robustly, though nonuniformly, detected only in tumor tissue (Fig. 4F).

CS GalNAc Transferase 1 and VCAN are More Altered in FLC Than in Most Other Cancer Types and Correlate with DNAJB1-PRKACA Levels
Next, we sought to compare the expression of CSGALNACT and VCAN in FLC with other cancer types. Specifically, we queried The Cancer Genome Atlas (TCGA) database, which houses RNA-seq data from 25 other cancer types. We found that the change in expression of CSGALNACT in FLC (relative to corresponding nonmalignant tissue) is second only to cholangiocarcinoma (CCA; Fig. 5A). Strikingly, the change in expression of VCAN is greatest in FLC, followed by CCA (Fig. 5B). Further analysis in an independent cohort revealed that VCAN and CSGALNACT are correlated (Fig. 5C). In addition, we found by qRT-PCR that the expression of DP correlates with both VCAN (Fig. 5D) and CSGALNACT (Fig. 5E).

VCAN is Expressed in FLC Transformed Epithelial and Tumor-associated, Activated Stellate Cells in FLC
To identify which cells are likely responsible for VCAN production and secretion, we performed scATAC-seq on NML, primary FLC tumor, and metastatic FLC tumor samples (n = 3). We used a previously described nuclei isolation protocol (34) and obtained data on nearly 9,500 nuclei total. After data analysis with ArchR (ref. 41; Materials and Methods), nonlinear dimensionality reduction via Uniform Manifold Approximation and Projection (UMAP) revealed eight different clusters (Fig. 6A). By analyzing open chromatin signal at established markers of human liver cell types (53), we assigned each cluster to a specific cell type ( Fig. 6A; Supplementary Fig. S4A-D).
We also analyzed open chromatin in the deleted region of chromosome 19 to identify the cells that likely harbor the deletion and therefore the DP fusion (Supplementary Fig. S5A and S5B). We then queried for ATAC signal associated with CSGAL-NACT and detected robust enrichment in the FLC primary and metastatic tumor transformed epithelial cell clusters (Fig. 6B). As expected, there is little to no signal for open chromatin at CSGALNACT in any nonmalignant cell types ( Fig. 6B and C). A similar analysis for VCAN revealed the strongest signal in tumor-associated activated stellate cells and secondmost in tumor transformed epithelial cells (Fig. 6D and E). These findings suggest that while CS synthesis (via CSGALNACT1) is likely exclusively taking place in tumor transformed epithelial cells, the primary CSAP in FLC, VCAN, is produced and secreted from both activated stellate cells as well as tumor transformed epithelial cells (Fig. 7).

Discussion
FLC is an aggressive liver cancer that lacks an effective chemotherapeutic remedy. There are several factors contributing to low survival rates in FLC, including vague manifestations, lack of comorbidities, and resistance to general therapeutics. FLC is genetically characterized by the DNAJB-PRKACA (DP) fusion, but efforts to identify specific inhibitors of DP, without targeting wildtype PRKACA, have been unsuccessful. In addition, while it is known that DP is sufficient for tumor initiation, it is unclear whether DP expression is essential for tumor maintenance, progression, and metastasis. It has been established in the study of other cancer types that the pericellular environment, including PGs, plays an important role in defining tumor behavior (12,15). However, to date, no study has investigated GAGs and PGs in FLC. In this study, we sought to bridge this knowledge gap.
The three major classes of GAGs are HA, HS, and CS (3). Of these, only HA is found as a free polymer generated by HA synthetase 1-3 (HAS1-3).
The expression levels of these enzymes were found to be extremely low in FLC and, therefore, they were not considered further. HS and CS chains are conjugated to proteins by a shared tetrasaccharide linker. The enzymes responsible for the synthesis of this linker exhibit robust expression in FLC but are unchanged relative to NML tissue. The decision by cells to generate HS or CS sidechains is dependent on stoichiometric competition between the enzymes N-acetylglucosaminyltransferase (EXTL), responsible for HS chains, and chondroitin GalNAc transferase (CSGALNACT), responsible for CS chains. We found that the ratio of CSGALNACT to EXTL levels is dramatically elevated in FLC, pointing to CS chains as the key component of the extracellular matrix in FLC.
A major innovation and strength of this study is the development and implementation of a highly sensitive method for quantifying HS and CS disaccharides in patient tissues. This novel assay confirmed that the alterations in expression of CS biosynthetic genes observed in FLC lead to dramatic changes in CS abundance. The quantity of CS in FLC tissue relative to NML was greater than 5.9-fold and the relative difference between CS and HS in tumor tissue was 4.3fold. In addition, this assay independently quantifies sulfated forms of CS and HS disaccharides. As HS abundance is unchanged in FLC tissue, it is not surprising that there are no significant differences in the seven sulfated forms of HS that we measured. The analysis of sulfated forms of CS showed that nonsulfated CS and two forms of monosulfated CS (CS di-4S and CS di-6S) are significantly increased in FLC tissue. The increased abundance of CS di-4S and CS di-6S is concordant with gene expression increases in associated chondroitin sulfotransferases (CHST/), again showing that changes in gene expression accurately correspond with changes in chemical abundance. It has been observed in HCC that increased expression of CHST/, which are functionally equivalent, are upregulated in metastatic samples (54), which may promote sustained Wnt signaling (55). One limitation of the quantitative analysis for the CS chains with di-2S4S should be noted. We were unable to synthesize 13 C-labeled di2S4S; therefore, the quantitation was completed using the relative quantitation method. However, this should not affect our conclusions, as the levels of di-2S4S are the same in FLC compared with NML tissues.
Given the abundant increase of CS in FLC tissue, and the high expression of the CSAP VCAN, we conjectured that the levels of CSGALNACT and VCAN would be correlated. Indeed, we found that there is a positive correlation between the two genes, and between either gene and DP. Consistent with this finding, in a previous report we had demonstrated that the expression of DP in an FLC cell model increases VCAN expression (5). We found in this study that the levels of VCAN are upregulated in FLC more than in any other cancer type for which expression data are publicly available through TCGA. CCA is the closest to FLC in terms of CSGALNACT and VCAN upregulation. Intriguingly, CHPF has been reported recently to promote CCA cell growth and invasive potential (56). In addition, CHSY has been reported to suppress apoptosis in colorectal cancer (57) and promote migration in HCC (58).  The expression and secretion of VCAN from activated stellate cells is a normal response to liver injury (60)(61)(62)(63). The data generated in this study suggest AACRJournals.org Cancer Res Commun; 2(7) July 2022  that FLC cells upregulate CSGALNACT and VCAN in a DP-dependent manner and begin secreting CS-VCAN PG into the extracellular matrix. The increased accumulation of VCAN may sequester higher concentrations of growth factors, or increase mechanical tension, and induce the activation of quiescent stellate cells. Upon activation, these stellate cells proliferate and secrete VCAN as a normal response to a perceived injury. VCAN is a highly modular protein containing four distinct domains (G1, GAGα, GAGβ, and G3). The G1 and G3 domains govern direct interactions with extracellular matrix components and cell surfaces, such as with HA and EGF receptor, respectively. The GAGα and GAGβ domains contain sites for CS chain conjugation. Through alternative splicing, six distinct isoforms, V0-4 and versikine (a secreted version), have been described. Only the V0 isoform contains all four domains and V1 and V2 contain GAGβ or GAGα, respectively. The V3, V4, and versikine isoforms are smaller peptides and contain little-to-no sites for CS conjugation.
The CS containing isoforms V0, V1, and V2 may also differ across cell types in terms of degree of CS conjugation, elongation, and sulfation, all which affect interactions with soluble factors. Because of these variables, VCAN can promote pleiotropic downstream effects; therefore, determining the specific functions of VCAN in cancer is not trivial (64,65). Identifying which protein isoforms of VCAN are expressed in FLC tumor epithelial cells and activated stellate cells is a critical next step toward defining the role of VCAN in FLC progression.
The role for stellate cells to promote fibrosis and predispose the liver to cancer formation is well established (66) and multiple HS PGs have been implicated in this role, including syndecans (67)(68)(69)(70), glypicans (71)(72)(73), and even free HS disaccharides (74). Activated stellate cells can promote the formation of CCA (75), a characteristically desmoplastic tumor. Our finding that CSGALNACT and VCAN expression levels in FLC are most similar to that of CCA suggests potential mechanistic parallels between the two cancer types in terms of fibrosis. In addition, CS PGs, including VCAN and CD44, have been implicated in both hepatic fibrosis and HCC formation (31,(76)(77)(78). However, these findings in HCC suggest that stellate-mediated fibrosis precedes and contributes to cancer formation. Given that patients with FLC lack preceding fibrotic conditions, such as cirrhosis, the relationship between stellate activation, fibrosis, and cancer development in FLC and CCA may be fundamentally different relative to HCC. Further investigation is required to define the roles of VCAN in tumor proliferation and invasion, either by direct influence on tumor epithelial cells, or by indirect means such as communication with activated stellate cells to affect the ECM, or both. These follow-up studies may also reveal whether VCAN is compelling as a direct therapeutic target in FLC.
We have implemented several novel methods to provide the first highresolution analysis of PG biology in FLC tumors. Our findings motivate further investigation of VCAN in FLC progression. Future research may also study the effects of VCAN inhibitors on FLC cell drug resistance, growth and/or metastasis.