GD2 and its biosynthetic enzyme GD3 synthase promote tumorigenesis in prostate cancer by regulating cancer stem cell behavior

While better management of loco-regional prostate cancer (PC) has greatly improved survival, advanced PC remains a major cause of cancer deaths. Identification of novel targetable pathways that contribute to tumor progression in PC could open new therapeutic options. The di-ganglioside GD2 is a target of FDA-approved antibody therapies in neuroblastoma, but the role of GD2 in PC is unexplored. Here, we show that GD2 is expressed in a small subpopulation of PC cells in a subset of patients and a higher proportion of metastatic tumors. Variable levels of cell surface GD2 expression were seen on many PC cell lines, and the expression was highly upregulated by experimental induction of lineage progression or enzalutamide resistance in CRPC cell models. GD2high cell fraction was enriched upon growth of PC cells as tumorspheres and GD2high fraction was enriched in tumorsphere-forming ability. CRISPR-Cas9 knockout (KO) of the rate-limiting GD2 biosynthetic enzyme GD3 Synthase (GD3S) in GD2high CRPC cell models markedly impaired the in vitro oncogenic traits and growth as bone-implanted xenograft tumors and reduced the cancer stem cell and epithelial-mesenchymal transition marker expression. Our results support the potential role of GD3S and its product GD2 in promoting PC tumorigenesis by maintaining cancer stem cells and suggest the potential for GD2 targeting in advanced PC.


Prostate cancer (PC) patient tissue microarrays and immunohistochemical analysis
All methods and experimental protocols were carried out in accordance with relevant guidelines and regulations of Institutional Biosafety Committee of the University of Nebraska Medical Center.Tissue microarrays (TMAs) composed of 320 cases of paired normal prostate and prostate cancer Gleason grades (3, 4 and 5) and or of 45 cases of bone and visceral metastatic samples from Rapid Autopsy TMAs were obtained without any identifying patient information from the Prostate Cancer Biorepository Network (PCBN) repository under a materials transfer agreement.320 prostate specimens used for immunohistochemical analysis were radical prostatectomy samples selected from the surgical pathology files at the Johns Hopkins Department of Pathology with Institutional Review Board approval.45 cases of metastatic TMAs were obtained from patients who died of metastatic CRPC and signed written informed consent for a rapid autopsy to be performed within hours of death, under the aegis of the prostate cancer Donor Program at the University of Washington.The use of metastatic TMAs for IHC analysis was approved by the Institutional Review Board at the University of Washington.
TMAs were stained for GD2 by immunohistochemistry (IHC) as described previously 27 .Briefly, TMAs were deparaffinized in xylene, rehydrated in descending alcohols and heated in the antigen retrieval solution (vector, #H-3300) in a microwave to unmask the antigens.Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 20 min.The sections were rinsed in phosphate buffered saline (PBS) and blocked in Protein-Block buffer (DakoCytomation #0909) for 15 min.The sections were then stained with anti-GD2 antibody (2 μg/ml) overnight at 4 °C.After rinsing in TBST, the sections were incubated for 15 min with an anti-mouse secondary antibody conjugated to a dextran-labeled polymer and HRP (DakoCytomation K4007), followed by incubation in DAB solution (DakoCytomation DAB # K4007, Solution 3a, b) for 5 min.The sections were counter-stained with hematoxylin and mounted under cover glasses using a xylene-based mounting medium.GD2 scoring was evaluated by a certified pathologist (SL) and staining scores were assigned as: 0 (negative), 1 (low positive), 2 (moderate positive) and 3 (strong positive).Four cores per case was analyzed for primary tumors and three cores per case for metastasis.Histoscore was calculated by multiplying the staining intensity by % cells staining.Histoscore distributions were assigned as follows: 0-5, negative; 5-25, low; 25-50, moderate; and > 50, strong positive.Histoscore 5 was used as a cut off.

Analysis of ST8SIA1 mRNA expression in transcriptomic data of CRPC patient-derived xenograft (PDX) tumors and organoid models
ST8SIA1 mRNA expression was analyzed using publicly available transcriptomic data on CRPC PDX and organoid models published by Tang et al. 26 (GEO accession GSE199190).As per the author classification, the CRPCs were grouped into 4 groups: Group 1, Androgen Receptor dependent (CRPC-AR); Group 2, Wnt dependent (CRPC-WNT); Group 3, neuroendocrine (CRPC-NE); and Group 4, stem cell like (CRPC-SCL) were analyzed.Gene expression is presented as normalized transcripts per million (TPM).

Intra-tibial tumorigenic growth and IVIS imaging
This study was performed in accordance with relevant guidelines and regulations.All animal experiments were performed in accordance with the ARRIVE guidelines with the approval of the UNMC Institutional Animal Care and Use Committee (IACUC; Protocol Approval # 21-004-06 FC). 4 × 10 4 (in 20 μl cold PBS) parental (control) or GD3S-KO (sgRNA1 and sgRNA2) RM-1 cells engineered with lentiviral tdTomato-luciferase or 1 × 10 5 (in 40 μl cold PBS) parental (control) or GDS3-KO (sgRNA1) 22Rv1 cells engineered with lentiviral mCherry-enhanced luciferase were injected into tibias of 8-weeks old castrated male C57BL/6 (for RM-1 model) or athymic nude (for 22Rv1 model) mice, respectively, and tumor growth was monitored by bioluminescence imaging .Mice were imaged weekly and followed until the control group reached pre-determined humane end points requiring euthanasia.At necropsy, hind limbs, lungs, spleen, and livers were harvested.For bioluminescence imaging, mice received 200 μl D-luciferin (15 mg/ml; Millipore Sigma #L9504) intraperitoneally 15 min before isoflurane anesthesia and were placed dorso-ventrally in the IVIS™ Imaging System (IVIS 2000).Images were acquired using the IVIS Spectrum CT and analyzed using the Living Image 4.4 software (PerkinElmer).The fold change in total Photon flux (p/s) over time was used to compare tumor growth between the groups.
RT 2 profiler PCR arrays mRNA was isolated from parental and GD3S-KO RM-1 cells using QuantiTech Reverse Transcription Kit (#205310).The expression of specific genes was quantitated by qPCR using the RT 2 Profiler PCR Array (PAMM-176ZA-6 RT 2 Profiler™ PCR Array for mouse cancer stem cells; catalog #330231) with QuantiTech SYBR Green PCR Kit (#204141) on an Applied Bioscience QuantStudio thermocycler.The analysis was performed using the GeneGlobe online application (geneglobe.qiagen.com)and the fold regulation of the genes was represented against the control in the heatmap and volcano plot.

Isolation of gangliosides, thin layer chromatography, and immunostaining
The isolation of gangliosides, thin layer chromatography and immunostaining were carried out using previously described protocol with minor modifications 30 .8 × 10 7 cells were trypsinized and resuspended in 2 ml of Chloroform: Methanol (at the ratio 2:1, v/v), and sonicated in an ultrasonic bath (Emerson Branson 1800) for 15 min at room temperature.The cells were centrifuged @ 2000 rpm for 5 min and supernatants were transferred to clean glass tube with PTFE-lined screw cap.Re-extraction of lipids was performed by adding 2 ml of Chloroform: Methanol (1:1, v/v) and sonicated again for 15 min.The cells were centrifuged again @ 2000 rpm for 5 min and supernatant transferred to clean glass tube.Final extraction of lipids was done by adding 2 ml of Chloroform: Isopropanol: Water (7:11:2 ratio) and sonication for 15 min.The cells were centrifuged again @ 2000 rpm for 5 min and all supernatants were pooled together into a clean glass tube.C18 solid phase extraction cartridge (Waters, SepPak Plus long C18, #WAT023635) was prepped by first washing with 3 mL of methanol, then with 3 ml of Chloroform: Methanol: Water (2:43:55, v/v/v).The pooled supernatant was loaded onto the washed C18 cartridge using the glass syringe and the flow-through collected and reloaded onto the column to optimize adsorption.This was followed by washing the column with 5 ml water to desalt.The gangliosides were eluted with 3 ml of methanol, collecting the eluate in a fresh glass tube and the solvent was removed using Rota-Vapor R-210.The sample was dissolved in 200ul methanol and were run for Thin Layer Chromatography (TLC).
The coated Silica Gel-60 TLC plates (Sigma #HX30882453) were pre-run in chloroform to eliminate neutral lipid and other contaminants that may interfere with the mobility of gangliosides.The samples were loaded on to the dried TLC plates using Hamilton Syringe 1 cm above the bottom and 1 cm apart from each other.The loaded samples were air dried and then run in the solvent Chloroform: Methanol: 0.2% CaCl 2 (5:4:1, v/v/v) [Have tested 55:45:10, 45:55:10, 60:35:10, 70:30:10, 80:20:10].The TLC plate after the run was allowed to air dry and visualized using Phosphomolybdic acid [12-Molybdophosphoric acid in Ethanol (10% v/v)]. 1 μl (1 mg/ mL conc) of Ganglioside, GD2 standard (Cat#25487, Cayman Chemical) was loaded as a positive control.For immunostaining, the TLC plate after the run was dip in 0.2% polyisobutyl-methacrylate Hexane: Chloroform (9:1, v/v) for 1 min with sample side up and dried.The TLC plate was blocked using 1% BSA in PBS for 30 min at room temperature followed by 3 washes with PBS (3 min each).The plate was then immuno-stained overnight with 0.5 mg/ml anti-GD2 antibody at 4 °C.The next day the plate was washed 3 times with PBS (3 min each) and stained with HRP conjugated secondary antibody at 1:500 dilution for 1 h.at room temperature.The plate was washed thrice with PBS and developed using the ECL substrate.

Statistical analysis
GraphPad Prism software (version 9) was used to perform the statistical analyses.The in vitro experimental data analysis used a two-tailed student's t test.A two-way ANOVA (mixed model) test was used to analyze the fold change in photon flux in xenograft studies.P values equal to or < 0.05 were considered significant.

Increased GD2 expression upon RB1 or TP53 knockdown in a CRPC cell line model
Given the GD2 overexpression in neuroectodermal lineage tumors 3 , and prior reports that CRPC-NE differentiation is associated with the acquisition of CSC features 31 , we surmised that GD2 overexpression might be a feature of CRPC upon experimental perturbations shown to promote neuroendocrine differentiation.RB1 or TP53 depletion in androgen receptor-driven CRPC (CRPC-AR) cell lines 32 or mouse models 33 combined with PTEN loss is known to promote NE trans-differentiation.Hence, we compared the CRPC-AR cell line LNCaP C4-2 (referred to as C4-2), which has a mutationally-inactivated PTEN 34 , with its stable TP53, RB1 or TP53/RB1 knockdown (KD) derivatives as previous studies in this model showed that TP53/RB1-KD cells had acquired NE differentiation 28 .qPCR analysis verified the expected TP53 or RB1-KD (Fig. 1B).Western blotting confirmed the knockdown of RB1 and TP53 in individual KD and DKD cells (Supplementary Fig. S1A).We validated a commercially available anti-GD2 antibody (clone 14.G2; Biolegend) by demonstrating its reactivity www.nature.com/scientificreports/by FACS and immunohistochemistry (IHC) against a known GD2 + neuroblastoma cell line SK-N-BE-2 35 and the lack of its reactivity with a GD2 -rhabdomyosarcoma cell line A204 36 (Supplementary Fig. S1B,C).Using this antibody, FACS staining revealed low cell surface GD2 expression on parental C4-2 cells, but the levels of GD2 and the size of the GD2 + subpopulation markedly increased in RB1-KD, TP53-KD or double-KD (DKD) C4-2 derivatives (Fig. 1C).Notably, the increase in GD2 levels on DKD was intermediate between that with individual KD of RB1 or TP53, the latter being higher than with RB1-KD (the MFI and % GD2 + cells are indicated inside FACS plots).IHC analyses confirmed the upregulation of GD2 in RB1-KD and TP53-KD cells compared to the parental C4-2 cells (Fig. 1D).Increased expression of GD2 upon TP53 or RB1-KD of C4-2 cells was associated with increased levels of the key GD2 biosynthesis enzymes GM3 Synthase (GM3S), GD3 synthase (GD3S) and GD2 synthase (GD2S) (Fig. 1E).
The GD3S enzyme (encoded by the ST8SIA1 gene) is rate-limiting for GD2 synthesis 2 .Consistent with our staining data, the CCLE-DepMap mRNA expression data available for ten PC cell lines showed the highest value for the GD2 high 22Rv1 and the second lowest value for the GD2 -PC3 cell line (Supplementary Fig. S3, upper panel).Analysis of transcriptomics data of organoids and PDX tumors recently used to classify CRPC into 4 subtypes, CRPC-AR (AR-driven), CRPC-NE (neuroendocrine), CRPC-WNT (WNT pathwaydriven) and CRPC-SCL (stem cell like) 26 , revealed high ST8SIA1 mRNA expression in GD2 high 22Rv1 and nearly undetectable levels in GD2 -LNCaP cell line (Supplementary Fig. S3, upper panel).Using the levels in these cell lines as reference, > 20% of CRPC organoids and PDX tumors showed higher ST8SIA1 expression (those above the red cutoff line corresponding to TPM values for LNCaP), and this was seen across the CRPC subtypes (Supplementary Fig. S3).Interestingly, the ST8SIA1 TPM values in a number of organoid models were substantially higher than those for 22Rv1(e.g., CRPC-AR samples MSK-PCa19, MSK-PCa22; CRPC-NE Table 1.Prostate cancer cell lines show % of GD2 on live cell FACS analysis using anti-GD2 staining. samples MSK-PCa10, MSK-PCa24; and CRPC-SCL sample MSK-PCa8).Thus, our results show that a subset of established PC cell lines, including examples of CRPC, express low to high GD2 levels on a variable fraction of cells, reminiscent of studies with breast cancer cell models 9,10 , and the ST8S1A1 expression in organoid and PDX models supported the potential of high GD2 expression in patient-derived PCs.

GD2 expression is seen on a small subset of tumor cells in primary PC patient tissues and a higher proportion of metastatic lesions
To assess if GD2 expression on a subpopulation of cells in PC cell lines could be extended to human PC, we used tissue microarrays (TMAs) from two PC patient cohorts (provided by the Prostate Cancer Biorepository Network; PCBN).IHC staining of a 320-sample TMA of paired normal prostate tissues and PC samples (Gleason scores of 3-5) demonstrated a statistically significant overexpression of GD2 on primary PCs compared to paired normal prostate tissue (Fig. 2A-D); unlike uniform GD2 + neuroblastomas, but similar to triple-negative breast cancers, only a small subset of tumor cells in PC samples was GD2 + .Overall, 19% of primary PCs scored GD2 + based on a histoscore cutoff of < 5 (Fig. 2E).Notably, IHC analysis of a metastatic PC TMA (the 45-Case Bone and Visceral Metastasis from Rapid Autopsy TMA from PCBN) showed GD2 + staining in about twice the percentage of metastatic lesions compared to primary tumors (Fig. 2E).The histoscore distribution analysis showed that metastatic tumors exhibited higher GD2 expression (Fig. 2F,G).These results indicate that GD2-expression is a feature of a subpopulation of tumor cells in a subset of PC patients, especially in metastases.
Previous studies have shown that GD2-expressing cell fraction is enriched in tumorigenic behaviors 9 .We therefore used FACS-sorting to purify the GD2 high and GD2 low populations of 22Rv1 and RM-1 cell lines as these cell lines harbor both GD2 high and GD2 low cell subpopulations (Table 1).Consistent with previous studies demonstrating that GD2 expression on cell lines is dynamic 41 , the FACS-sorted populations drifted towards a mix of GD2 high and GD2 low populations upon continued 2D or 3D culture (Fig. 3D).Notably, FACS analysis revealed a significant enrichment for the GD2 high population during tumorsphere growth vs 2-D culture in both RM-1 (72% and 89% at days 3 and 5 in 3D, and 56% and 59% at 3 and 5 days in 2D, vs. 56% on Day 0; p < 0.001) and 22Rv1 (87% and 94% at days 3 and 5 in 3D, and 18% and 21% at 3 and 5 days in 2D, vs. 16% on Day 0; p < 0.001) cell lines (Fig. 3E).A similar, time-dependent, increase in the GD2 high population was observed upon tumorsphere vs. 2D growth of C4-2 (GD2 low ) and its RB1-KD (GD2 high ) derivative (Supplementary Fig. S4).Despite the dynamic nature of GD2 expression, the GD2 high fractions of both 22Rv1 and RM-1 cell lines yielded significantly more tumor-spheres (~ 22/field vs. 16/field for GD2 high and GD2 low RM-1, and 32/field vs. 11/field for GD2 high and GD2 low 22Rv1; p < 0.01 and < 0.001, respectively) (Fig. 3F) indicating a higher tumorsphere-forming ability of GD2 high subpopulation in PC cell lines.Dual FACS staining of RM-1 and 22Rv1 cell lines grown as tumorspheres for CD49f.and GD2 demonstrated that a majority (60.9% for RM-1 and 58.8% for 22Rv1) of GD2 high cells were within the CD49f high fraction (Fig. 3G).These results suggested that GD2 high fraction defines a cell population with higher oncogenic potential and potentially the CSC-like behavior.

GD3 synthase, the rate-limiting enzyme in GD2 biosynthesis, is required for in vitro oncogenic attributes of GD2 high CRPC cell lines
In view of our results above, we hypothesized that the GD2 + cell subpopulation is the major driver of the oncogenic attributes of PCs.To test this hypothesis, we targeted the rate-limiting GD2 biosynthesis enzyme GD3S 2 for CRISPR-Cas9 knockout (KO), using a single-vector Cas9/sgRNA expression system, in GD2-overexpressing mouse (RM-1) and human (22Rv1) CRPC cell lines and obtained clonal lines by serial limiting dilution cloning.Western blotting confirmed the loss of GD3S expression in GD3S sgRNA-targeted derivatives (Fig. 4A, B).These GD3S-KO clones indeed lacked the cell surface GD2 expression by FACS analysis (Fig. 4C, D).We also analyzed the parental vs. GD3S-KO RM-1 and 22Rv1 cell lines together with GD2 + 9464D and GD2 -975A2 mouse neuroblastoma cell lines 41 by thin layer chromatography (TLC) and TLC/immunoblotting, in which lipid extracts of cells were resolved by TLC next to the purified GD2 standard, followed by either chemical staining to visualize the separated sphingolipid species or immunoblotted with the 14G2a antibody, respectively.Based on pilot studies using various ratios of the chloroform/methanol/0.2%CaCl 2 solvent system used in prior studies [42][43][44] , we resolved the sphingolipid extracts of cell lines using the 50:40:10 ratio of chloroform/methanol/0.2%CaCl 2 as these were optimal to resolve GD2 and GD3 species from other lipids.Phosphomolybdic acid (PMA) staining of the resolved species on the TLC plate showed lipid species comigrating with standard GD2 in RM-1, 22Rv1 and the positive control 9464D cells, while these species were absent in the GD3S-KO cell lines and the GD2 negative 975A2 cells (Supplementary Fig. S5A).Notably, 14G2a immunoblotting of the TLC-resolved sphingolipid species showed strong reactivity with the GD2 standard, as expected; additionally, the 14G2a antibody detected the lipid species comigrating with standard GD2 in parental RM-1 or 22Rv1 and in the positive control cell line 9464D (corresponding to the comigrating species detected by chemical staining) but not in GD3S-KO RM-1 or 22Rv1 cell extracts or in the negative control cell line 975A2 (Supplementary Fig. S5B).These results further confirm the reactivity of 14G2a with GD2 and the absence of its expression upon GD3S-KO.As GD3 is the immediate product of GD3S activity and a precursor of GD2, we also assessed the levels of GD3 in panels of PC cell lines (Supplementary Fig. S6).In addition, parental vs. GD3S-KO CRPC cell lines RM-1 and 22Rv1 were assessed for expression of GD3 using FACS.Relatively low to modest levels of GD3 were seen on parental cells and this expression was eliminated in KO cells (Supplementary Fig. S7).Thus, GD3S-KO effectively eliminated the expression of GD2 (and GD3) on CRPC cell lines.We also generated GD3S-KO derivative of the enzalutamide-resistant C4-2BER cell line, which has been reported to express NE markers 28 and expresses high GD2 levels compared to its parental cell line (Supplementary Fig. S2C and S8A).GD3S-KO eliminated the expression of GD2 in this model as well (Supplementary Fig. S8B).Furthermore, GD3S-KO led to reduction in the expression of NE differentiation markers, neuron specific enolase (NSE), Enhancer of Zeste homolog 2 (EZH2) and chromogranin A (CHGA) compared to C4-2BER cells, with levels in GD3S-KO cells comparable to those in GD2 low C4-2B cells (Supplementary Fig. S8C).These results suggest GD2 expression may regulate NE differentiation.
Functional analyses of various oncogenic traits showed a significant impact of GD3S-KO.Using CellTiterGlo assays, the GD3S-KO RM-1 cells exhibited a fold-increase of ~ 51-fold compared to ~ 73-fold for control cells, while GD3S-KO 22Rv1 cells showed a ~ 18-fold-increase compared to a ~ 28-fold increase for parental cells, with the differences being statistically significant (p < 0.05) (Fig. 4E,F).In cell migration assays, GD3S-KO RM1 and 22Rv1 cell lines exhibited a 55% or 65% lower migration, respectively, relative to their controls (p < 0.001) (Fig. 4G).A similar impairment of transwell invasion ability was seen upon GD3S-KO, with the GD3S-KO RM-1 and 22Rv1 cell showing 67.5% and 73% lower invasion, respectively, relative to their parental cell lines (p < 0.001) (Fig. 4H).Tumorsphere assays showed that GD3S-KO RM-1 and 22Rv1 cell lines exhibited 88% or 90% reduced tumorsphere forming abilities compared to their parental non-targeted cells (p < 0.001) (Fig. 5A), supporting the importance of GD3S and its ganglioside products in promoting the CSC behavior of CRPCs.Further supporting this notion, FACS analysis for the proportion of CD49f hi cells, a marker associated with PC CSCs 45 , showed a reduction from 47 to 22% for GD3S-KO RM-1 (p < 0.01) and 53% to 39% for GD3S-KO 22Rv1(p < 0.05), relative to their parental lines (Fig. 5B).To further evaluate the potential linkage of GD3S and GD2 expression with CSC characteristics, we analyzed the mRNA expression levels of 84 mouse genes included in a commercial CSC signature panel (RT 2 profiler array) using quantitative PCR (qPCR) analysis of control vs. GD3S-KO RM-1 cells.The represented genes correspond to those associated with CSCs as well as Epithelial Mesenchymal Transition (EMT), given the strong mechanistic linkage of EMT and CSC behaviors 46 .Volcano plot analysis revealed multiple genes in the array to be either down-regulated or upregulated in the GD3S-KO derivative vs the control Figure 3. GD2 overexpression promotes the in vitro oncogenic traits: (A) Analysis of cell proliferation.The parental C4-2 and its TP53-KD or RB1-KD or TP53/RB-KD (DKD) cells and C4-2B and its enzalutamide resistant variant C4-2BER were plated in 96-well plates and cell proliferation was assessed on the indicated days using the CellTiter-Glo luminescent cell viability assay.The Y-axis represents the fold increase in relative luminescence units (RLU) relative to Day 1. n = 3 with six replicates each (B) Analysis of cell migration in indicated cell lines using 20,000 cells as input.Left, representative images.Right, quantification.n = 3 with three replicates each.(C) Analysis of tumorsphere forming ability.The indicated cell lines were plated in 24-well ultra-low attachment plates in presence of 4% Matrigel (upper and middle panels) and in absence of Matrigel.(lowerpanel) and images were obtained on Day 7.Left, representative images; scale bar, 1000 µm.Right, quantified tumorsphere numbers per 4X microscopic field.n = 3 with three replicates each and 6 images per well (D) GD2 high and GD2 low population were FACS sorted in RM-1 and 22Rv1 cells and grown as 2D and tumorspheres.After Day 5, the cells were reanalyzed for GD2 fractions in both GD2 high and GD2 low post sorted cells.In both tumorspheres and 2D culture the GD2 low cells re-expressed GD2.(E) Enrichment of GD2 high cells in tumorspheres of CRPC cell lines.The indicated cell lines were cultured in 2D or were seeded in tumorsphere cultures.Cells harvested at the indicated times were analyzed for cell surface GD2 expression (vs.isotype control) at the indicated times.% GD2 + cells and MFI are indicated.Left, representative FACS images; Right, quantitation of data.two-way ANOVA test.(F) GD2 high fraction of CRPC cell lines is enriched for tumorsphere-forming ability.Constitutively GD2-expressing RM-1 and 22Rv1 CRPC cell lines were FACS sorted for GD2 high and GD2 low populations, grown on 2D overnight and plated at 20,000 cells per well of 24-well ultra-low attachment plates in tumorsphere media containing 4% Matrigel.Tumorspheres were imaged on Day 5. Left, representative images; scale bar, 2000 µm.Right, Quantification of tumorspheres (> 250 µm) per 2× magnification field.n = 3 with three replicates each and 6 images per well.(G) FACS analysis of tumorspheregrown RM-1 and 22Rv1 cell lines after anti-GD2 and anti-CD49f double-staining shows a majority of GD2 + cells to be CD49f hi % GD2 and/or CD49f +/− populations are shown in the respective quadrants.Data represented in all experiments are mean ± SEM, unpaired t-test; ns , not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.◂ RM-1 cells (Fig. 5C).As presented in a heatmap (Fig. 5D), we observed a significant downregulation of 17 genes including Notch1, Stat3, Sox2, Pou5f1(Oct4), Mycn, Lin28b, Snai1, Zeb1, Epcam, Pecam1 and upregulation of 22 genes such as CD24a, Mertk, Slug, Foxa2, CD44, Aldha1a, Klf4 and Myc in GD3S-KO cells (Fig. 5D).Western blotting analysis validated the qPCR-observed downregulation of Snail, ZEB1, Lin28b, Notch1, Stat3 (⍺, β), SOX2 and the observed upregulation of Slug in GD3S-KO RM-1 and 22Rv1 cells vs. their control cells (Fig. 5E).Additional Western blot analysis of CSC/EMT markers SOX9 and N-cadherin revealed their downregulation in GD3S-KO RM-1 and 22Rv1 cells vs. their control cells (Fig. 5E).Collectively, these results are consistent with a conclusion that the GD2 high fraction in CRPC models defines a CSC-like population.

GD3S-KO impairs the tumorigenic ability of GD2 high CRPC cell lines
To assess if the marked reduction in the in vitro pro-oncogenic traits of PC cell lines upon GD3S-KO translates into impaired tumorigenesis in vivo, we used lentiviral infection to introduce a tdTomato-luciferase dual reporter into control and GD3S-KO RM-1 cell lines (the latter with two distinct sgRNAs).The tdTomato-high fractions were enriched by FACS-sorting and stable cell lines selected.We implanted the reporter-bearing control and GD3S-KO RM-1 cells in the tibias of castrated syngeneic C57BL/6 mice and monitored tumor growth (5 mice/group) over time by bioluminescence imaging.While control RM-1 cell implants generated tumors that showed a time-dependent increase in log10 photon flux in IVIS imaging that peaked at 512-fold increase at the endpoint compared to day 7, both of the GD3S-KO RM-1 cell implanted groups generated significantly smaller tumors whose average peak photon flux was 40-fold (sgRNA1) and 20-fold (sgRNA2) lower compared to the control group (p < 0.01 for both KO groups relative to control) (Fig. 6A,B).Morphometric analysis of tibial bone by micro-CT scanning showed reduced bone destruction by implants of GD3S-KO RM-1 cells relative to control as demonstrated by quantification of the bone volume (38.85% and 38.86% for sgRNA1 and sgRNA2 groups, respectively, relative to 30.5% in control; p values for both < 0.05), trabecular thickness (thickness of 141mm and 144 mm for sgRNA1 and sgRNA2 groups, respectively, relative to 127 mm in control; p values for both < 0.05), trabecular number (trabecular number per mm of 0.00288 and 0.00278 for sgRNA1 and sgRNA2 groups, respectively, relative to 0.0023 in control; p values of < 0.01 and < 0.05 for sgRNA1 and sgRNA2 groups, respectively) and separation (516mm and 527mm for sgRNA1 and sgRNA2 groups, respectively, relative to 470 mm in control; p values for both < 0.05) (Fig. 6C-G).To confirm the impact of GD3S-KO to impair the ability of implanted PC cells to form tumors in vivo, we further implanted the mCherry-luciferase expressing parental or GD3S-KO 22Rv1 cells intra-tibially in nude mice and analyzed these by IVIS imaging.Compared to a 15,984-fold increase in Log10 photon flux relative to time 0 for the control group, the GD3S-KO cell implants showed an increase of only 397.5-fold in peak Log10 photon flux (p < 0.001) (Fig. 6H,I).Using IVIS, we did not observe distal metastases within the time of observation used in our studies (although some animals showed bioluminescent signals at the initial observation time point) (Fig. 6H).Together, these analyses demonstrate that GD3S-KO in CRPC cell line models significantly impairs their ability to form tumors when implanted in bone and to induce bone destruction.

Discussion
Despite improvements in diagnosis and management of early PC, progression to CRPC and metastatic disease represent lethal transitions responsible for high death burden from PC. Targetable molecular pathways that mark or contribute to these lethal states can open new therapeutic avenues in PC.Here, we use PC patient-derived tumor tissue analyses and cell line-based mechanistic approaches to demonstrate that the di-ganglioside GD2 marks a small but functionally-critical tumor cell subpopulation in PC with enrichment in more advanced disease states.Our studies using CRPC cell models establish that GD2 defines a cancer stem cell-like subpopulation that contributes to oncogenic drive, thus raising the prospect of GD2 targeting in CRPC using clinically approved therapeutic agents.
While expression and functional roles of GD2 are established in a number of cancers, with GD2 targeting with antibodies now approved for therapy of neuroblastoma patients 8 , only few studies have examined the expression of GD2 in PC and its roles are not known.No prior studies have examined GD2 expression in PC Figure 4. GD3S knockout in CRPC cell lines impairs the in vitro tumorigenic and pro-metastatic traits.(A-D) Generation of GD3S knockout of RM-1 and 22Rv1 CRPC cell lines.Cells were transduced with lentiviral All-in-One CRISPR/Cas9 constructs and stable clones analyzed by Western blotting with anti-GD3S antibody; Hsc70, loading control.Known GD2 + (9464D) and GD2 -(975A2) mouse neuroblastoma cell lines were used as controls.(C, D) Complete loss of cell surface GD2 expression in GD3S-KO RM-1 and 22Rv1 clones.The parental cell lines and their GD3S-KO clones were live-cell stained with anti-GD2 (blue) or isotype control (red) antibody followed by FACS analysis.FACS plots indicate mean fluorescence intensity (MFI) on X-axis vs. Events Normalized to Mode on Y-axis.% positive cells and MFIs are indicated in FACS plots.(E, F) Reduced CRPC cell proliferation upon GD3S-KO.Three clones of parental and GD3S-KO cell lines maintained separately were mixed plated at 1,000 cells per well in 96-well plates and live cells quantified at the indicated times using the CellTiter-Glo cell viability assay.The Y-axis represents the fold increase in relative luminescence units (RLU) relative to Day 1. n = 3 with six replicates each.(G, H) Reduced CRPC cell migration (G) and invasion (H) upon GD3S-KO.20,000 parental or GD3S-KO RM-1 or 22Rv1 cells were plated in low serum medium in top chambers of transwell chambers without (migration) or with (invasion) Matrigel coating and allowed to migrate or invade for 16 h towards serum-containing medium in the bottom chambers.Left, representative images.Right, quantification.n = 3 with three replicates each and 6 images per well.All Data represents mean ± SEM with unpaired t test; **, P < 0.01; ***, P < 0.001, ***p < 0.001.tissues at a histological level but limited prior studies primarily using biochemical fractionation of glycolipids [22][23][24] are consistent with our findings.The latter studies utilized a small number of normal or tumor tissues with biochemical analyses of GD2 expression which did not provide an assessment of what proportion of PC patients express GD2, and whether GD2 was uniformly expressed or in a heterogenous pattern.Our analyses using immunohistochemical staining (Fig. 2) therefore provide unique new insights by demonstrating that GD2 is expressed on a larger proportion of tumors versus the normal prostatic tissue, and importantly that only a small subset of cells in patient tumors express GD2.Notably, a significantly higher proportion of metastatic tumors harbored such cells (Fig. 2E).The GD2 expression pattern in PC is reminiscent of non-neuroectodermal tumors like breast cancer with a small GD2 + tumor cell subpopulation 9 , with GD2 marking the rare stem cell population in normal tissues and tumors 47 .
Our studies using cell line models and FACS analyses (Table 1, Supplementary Fig. S2) establish that similar to other well-studied tumor models, that GD2 is expressed at the cell surface of tumor cells.While even GD2 + cell line models showed a non-homogenous GD2 expression, similar to breast and other non-neuroectodermal tumor systems 9 , the GD2 high and GD2 low populations appear to be in a dynamic equilibrium, as seen by the reemergence of mixed GD2 high /GD2 low populations when sorted GD2 high or GD2 low subsets were cultured in vitro (Supplementary Fig. S4), similar to that described in other tumor systems 41 .Whether GD2 expression in vivo is similarly dynamic is currently unknown, but potentially significant to explore in the future.In this regard, dramatic induction/elevation of GD2 expression under scenarios of significance to the evolution of CRPC, including experimentally promoting lineage plasticity of AR-driven C4-2 cells by TP53 or RB1-KD (Fig. 1C,D), or when GD2 low C4-2B cells were made enzalutamide-resistant (Table 1, Supplementary Fig. S2), suggest that GD2 may be functionally important in more advanced stages of PC, a suggestion consistent with our finding of high GD3S (St8SIA1) mRNA levels a subset of CRPC PDX and tumor-derived organoid models (Supplementary Fig. S3) represented in publicly-available transcriptomic data 26 .
Notably, the IHC-based GD2 staining in PC tissues (Fig. 2A-C) as well as cell lines (Fig. 1D and Supplementary Fig. S1C) was predominantly intracellular (cytoplasmic) in contrast with the plasma membrane staining of cell lines analyzed by FACS (Fig. 3D,E; Supplementary Fig. S2).Published IHC studies showed a similar patten of GD2, described as perinuclear granular "Golgi-like" pattern, in neuroblastoma 48 and breast cancer tissues 49 .It is likely that the difference reflects the use of live cells for FACS analysis (precluding any staining of intracellular pools of GD2) while IHC is performed on fixed and permeabilized tissue sections where intracellular GD2 would be accessible to the staining antibody.
That GD2 expression on only a subset of tumor cells was nonetheless critical for tumorigenesis (Fig. 6) is reminiscent of studies in breast and other cancers where depletion of GD2 by genetic or pharmacologic downregulation of GD3S expression 11,50 or by immunologic targeting of cell surface GD2 12 led to impaired tumorigenesis.In these previous studies, GD2 expression marked the CSC population, thus accounting for GD2's role in multiple tumorigenic traits.Strong co-staining of GD2 with the CSC marker, high CD49f, enrichment of the GD2 + (Fig. 3G) and CD49f hi (CSC) populations (Fig. 5B) in tumorspheres, the requirements of GD3S for tumorsphere growth (Fig. 5A) and alterations in CSC and EMT-related signature gene expression upon GD3S-KO (Fig. 5C-E) are consistent with this idea.However, more in-depth analyses of the GD2 high and GD2 low subpopulations of PC models, including PDX and organoid models expressing GD2, examining their tumor-initiation and maintenance capabilities under limiting dilution conditions 9 , resistance to conventional therapies (such as chemotherapy) 20 and other CSC behaviors will be needed to firmly establish that GD2 + subpopulation in PCs represents CSCs.
Since our current assignment of a functional role to GD2 relied on GD3S-KO, similar to GD3S depletion approaches employed by others 11,50 , additional studies are needed to formally rule out a role of GD3, the other GD3S product and precursor to GD2 2 .Although the surface GD3 levels on the two cell models we targeted for GD3S-KO were relatively modest (Supplementary Fig. S6 and S7), further immunological targeting of GD2 and/ or GD2S deletion should elucidate the functional role of GD2 versus GD3 in PC tumorigenesis.
In our studies, we primarily utilized the antibody staining to demonstrate the expression of GD2 in cell lines and patient tumors.While this approach is well-established, and we further authenticated the antibody used (14G2a) in our studies (Supplementary Fig. S1), it is known that this antibody may also recognize modified forms of GD2, such as O-Acetyl-GD2 51 , that are expressed in some tumors.In limited analyses, using TLC separation of extracted lipids and standard GD2, we found the 14G2a to recognize a sphingolipid species comigrating with Reduced CRPC tumorsphere forming ability upon GD3S-KO.The indicated cell lines were plated in 24-well ultra-low attachment plates in 4% Matrigel and images were obtained on Day 7. Left, representative images; scale bar, 1000 µm.Right, quantified tumorsphere numbers per 4X microscopic field.n = 3 with three replicates each and 6 images per well (B) Reduced expression of CSCs markers in GD3S-KO CRPC cell lines.The indicated cell lines were subjected to live cell staining followed by Left, representative FACS plots display CD49f.staining (MFI's) on X-axis and forward scatter on Y-axis.Right, quantitation of % of CD49f.-highpopulation.n = 3. (C) Volcano plot of the mouse cancer stem cell RT 2 profiler quantitative PCR array analysis of RM-1 controls vs. GD3S-KO cells.X-axis, log fold change; Y-axis, -log p-value.Green dots represent the downregulated genes and red dots display the upregulated one with a log1.5-foldchange cut-off.(D) The heatmap shows the significantly upregulated (red) and downregulated (green) genes in RM-1 GD3S-KO cells compared to control cells.; n = 3 independent experiments, p < 0.05 is deemed significant.(E) Reduced expression of cancer stem cells and epithelial-mesenchymal transition (EMT) markers in GD3S-KO CRPC cell lines.Lysates of the parental vs. GD3S-KO RM-1 or 22Rv1 cell lines were subjected to western blotting with antibodies against the indicated proteins, with Hsc70 or β-actin used as loading controls.Densitometric quantification is shown on top of each blot.Data represents mean ± SEM, unpaired t test; *p < 0.05, **p < 0.01, ***p < 0.001.authentic GD2, and loss of this species upon GD3S-KO (Supplementary Fig. S5).However, further biochemical validation using TLC and/or Mass Spectrometry methods will be needed to establish that GD2 expression we define here in PC represents GD2 and not its modified forms.This is particularly important for future analyses of tumor tissues to avoid possible false positive/negative results as the tissue fixation and other processing could affect how the GD2 antigen is displayed in the samples.Collectively, our novel findings reveal that GD2 overexpression is a feature of a large subset of PCs, that GD2 may be further induced during transitions associated with PC progression, and that the GD2 biosynthetic machinery enzyme GD3S is essential for efficient tumorigenesis in CRPC models.Our findings raise the prospect of anti-GD2 targeting in advanced PC.

Figure 2 .
Figure 2. GD2 expression on a subset of tumor cells in primary prostate cancer tissue with higher expression in metastatic lesions.Tissue microarrays (TMAs) composed of paired normal prostate and prostate cancer Gleason grades (3, 4 and 5) (n = 320) or 45 cases of bone and visceral metastatic samples (both obtained from the Prostate Cancer Biorepository Network) were analyzed by IHC staining for GD2.Histoscores were calculated by multiplying the staining intensity (0, negative; 1, low; 2, moderate positive; 3, strong positive) by the % tumor cells staining positive.(A, B) Representative examples of negative, low, moderate, and high GD2 staining in (A) primary, (B) bone metastatic and (C) visceral metastatic tumor samples.(D) Scattered plot shows histoscore distribution of primary tumors and normal prostate.GD2 expression is significantly higher in primary tumors than normal prostate.Data represents 34 cases of GD2 expression in tumors vs 24 in normal tissue, considering histoscore > 25 as positive.Mann-Whitney unpaired t-test used as statistical analysis; *, P < 0.05.(E) Pie charts displaying the % of cases that were GD2 + or GD2 -in primary and metastatic tumors samples.Histoscores of 0-5 were considered negative while scores higher than 5 were considered positive.(F, G) Frequency distribution plots histoscores plotted against (F) percentage of patients and (G) number of patients.The indicated histoscore grouping are: 0-5 (negative); 5-25 (low), 25-50 (moderate); and > 50 (strong). https://doi.org/10.1038/s41598-024-60052-3www.nature.com/scientificreports/

Figure 5 .
Figure5.Impairment of cancer stem cell (CSC)-associated traits in CRPC cell lines upon GD3S-KO.(A) Reduced CRPC tumorsphere forming ability upon GD3S-KO.The indicated cell lines were plated in 24-well ultra-low attachment plates in 4% Matrigel and images were obtained on Day 7. Left, representative images; scale bar, 1000 µm.Right, quantified tumorsphere numbers per 4X microscopic field.n = 3 with three replicates each and 6 images per well (B) Reduced expression of CSCs markers in GD3S-KO CRPC cell lines.The indicated cell lines were subjected to live cell staining followed by Left, representative FACS plots display CD49f.staining (MFI's) on X-axis and forward scatter on Y-axis.Right, quantitation of % of CD49f.-highpopulation.n = 3. (C) Volcano plot of the mouse cancer stem cell RT 2 profiler quantitative PCR array analysis of RM-1 controls vs. GD3S-KO cells.X-axis, log fold change; Y-axis, -log p-value.Green dots represent the downregulated genes and red dots display the upregulated one with a log1.5-foldchange cut-off.(D) The heatmap shows the significantly upregulated (red) and downregulated (green) genes in RM-1 GD3S-KO cells compared to control cells.; n = 3 independent experiments, p < 0.05 is deemed significant.(E) Reduced expression of cancer stem cells and epithelial-mesenchymal transition (EMT) markers in GD3S-KO CRPC cell lines.Lysates of the parental vs. GD3S-KO RM-1 or 22Rv1 cell lines were subjected to western blotting with antibodies against the indicated proteins, with Hsc70 or β-actin used as loading controls.Densitometric quantification is shown on top of each blot.Data represents mean ± SEM, unpaired t test; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6 .
Figure 6.Impairment of in vivo bone-implanted CRPC tumorigenesis by GD3S-KO.(A) Parental (Control) RM-1 cells or their edited versions carrying sgRNA1 or sgRNA2 (GD3S-KO) were engineered with tdTomato-luciferase and 4 × 10 4 cells injected in tibias of castrated male C57/BL6 mice (8/group) and primary tumor growth was monitored by bioluminescence imaging at the indicated time points in mice (5/group) with detectable bioluminescent signals on day 8. (B) Bioluminescence signals of RM-1 tumors over time are shown as log fold-change in photon flux over the time.Data are mean ± SEM, n = 5; Two-way ANOVA (mixed model); *p < 0.05.(C) Micro-CT scanned images of tibias isolated from mice implanted with the indicated RM-1 cell lines.μCT analyses of the trabecular bone was used to calculate the: (D) bone volume fraction (BV/TV%); (E) trabecular thickness (µm); (F) trabecular number (per µm); and (G) trabecular separation (µm).Data represent mean ± SEM, with unpaired t-test used for statistical analysis; *p < 0.05 and ** p < 0.01.(H) Impairment of tumorigenesis of 22Rv1 CRPC cells by GD3S-KO. 1 × 10 5 control or GD3S-KO (sgRNA1) 22Rv1 cells engineered with mCherry-luciferase were injected in tibias of castrated male athymic nude mice (6/group) and tumor growth was monitored by bioluminescence imaging in mice (5/group) with detectable bioluminescent signals on day.(I) Bioluminescence signals of 22Rv1 xenografts over time are shown as log fold-change in photon flux over time.Data are mean ± SEM, n = 5, with analysis by Two-way ANOVA (mixed model); **p < 0.01.