Galectin-1 Modulates Glycolysis via a GM1-Galactose-Dependent Pathway to Promote Hyperthermia Resistance in Hepatocellular Carcinoma

Abstract

Background and Aims:

Thermal ablation is the standard of care treatment modality with curative intent for early-stage non-resectable hepatocellular carcinoma (HCC), but a durable response is limited—with up to 40% of HCC patients eventually experiencing local recurrence on post-treatment surveillance. While thermal ablation has been established to cause immediate cell death in the center of the thermal ablation zone, its metabolic impact in the peri-ablational region remains unclear. We aimed to elucidate the metabolic mechanism by which Galectin-1 (Gal-1) promotes thermal-ablation-induced hyperthermia resistance in HCC and demonstrate the therapeutic potential of inhibiting Gal-1 in combination with thermal ablation in vivo.

Approach and Results:

Proteomic analysis was performed using an untargeted approach on pre-ablation formalin-fixed paraffin-embedded (FFPE) biopsy specimens of thermal ablation responders (n=32) and nonresponders (n=23). Gal-1 was found to be overexpressed in thermal ablation nonresponders compared to responders. Moreover, HCC with Gal-1 overexpression demonstrated reduced sensitivity to hyperthermia in vitro and increased utilization of glycolysis and downstream TCA cycle under hyperthermia-induced stress. Gal-1-overexpressing HCC enhanced its metabolic utilization through Gal-1-facilitated GM1-ganglioside breakdown, producing galactose to increase the metabolic influxes into glycolysis and consequently the downstream TCA cycle. In vivo studies showed that inhibiting Gal-1 in combination with thermal ablation significantly reduced tumor size compared to either monotherapy thermal ablation or Gal-1 inhibition alone.

Conclusions:

Gal-1 can mediate hyperthermia resistance in HCC and can potentially be modulated as a therapeutic target to reduce rapid progression after thermal ablation.

Graphical Abstract:

III. INTRODUCTION

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the third leading cause of cancer-related death in the world.¹ Thermal ablation has historically been utilized with curative intent for early-stage, non-surgical HCC patients.² While the goal of thermal ablation is to treat the targeted HCC with adequate margins to achieve complete pathologic necrosis, there are select cases where there is rapid HCC progression after ablation, even in cases with technical success with adequate margins.^3,4

The rationale for rapid HCC progression lies in the fact that sublethal hyperthermia is created at the edges of the thermal-ablation zone.⁵ The center zone has been well characterized in thermal-dose models, and is usually associated with immediate cell death.⁶ However, this sublethal-hyperthermic environment is insufficient to eliminate peripheral tumor cells and can create a pro-tumorigenic milieu, exacerbating metabolic derangements associated with more aggressive HCC.⁵ These metabolic interactions can be amplified in imaging-occult-HCC types, such as in poorly-differentiated or macrotrabecular subtypes, when these are exposed to hyperthermic regions of the peri-ablational zone.⁷

Metabolic alterations within the tumor microenvironment is known to play a key role in post-ablation HCC-cell survival and progression,⁸ as rapid growth requires increased energy production, macromolecular biosynthesis, and maintenance of redox balance. However, in-depth analysis of early-stage HCC-tumor microenvironment and associated metabolic reprogramming, particularly under hyperthermic conditions, has not been well evaluated. Part of the reason for this knowledge gap is that studying the molecular aberrations of HCC requires clinical samples, which is currently not within the standard guidelines for HCC diagnosis.² HCC management guidelines for diagnosing HCC primarily rely on imaging alone, restricting the use of needle biopsy in only select-equivocal cases, rather than routine diagnosis.² Consequently, the underlying mechanisms behind early-stage-HCC post-ablation progression and associated-metabolic pathways remain understudied.

A potential target that has been implicated in HCC progression is Galectin-1 (Gal-1), an evolutionarily conserved, glycan-binding protein associated with cancer invasion and treatment resistance.^9–11 Gal-1 has been found to be overexpressed and modulate metabolism in other tumor types such as prostate, pancreas and brain.^11–13 Specifically, in glioma, Gal-1 functions as an intermediate-signaling activator for aerobic glycolysis¹³—a key ATP-producing mechanism conserved across many cancers, also known as Warburg effect.¹⁴ However, the direct metabolic role of Gal-1 has not yet been linked to post-treatment cancer metabolism, especially post-ablation in HCC. This study aimed to elucidate the mechanism by which Gal-1 mediated glycolysis and consequently the downstream TCA cycle to enable HCC cells to resist peri-ablational hyperthermic stress. Specifically, we characterized the role of Gal-1 in promoting the hydrolysis reaction between GM1 and catalytic enzyme β-galactosidase, ultimately creating galactose and subsequently metabolic influxes for glycolysis and TCA cycle.

To elucidate the mechanism and metabolic role of Gal-1, we first employed liquid chromatography-mass spectrometry (LC-MS) to analyze differential Gal-1 expression in a unique retrospective set of pre-ablation needle-biopsy samples from early-stage HCCs of patients subsequently identified as thermal-ablation responders and nonresponders. Subsequently, we demonstrated that Gal-1 modulation directly correlated with hyperthermia sensitivity and mediated the enhanced ability of HCC cells to utilize glycolysis and downstream TCA cycle in vitro, particularly under hyperthermic stress. Finally, these results were validated in an orthotopic murine model, where the combined approach of ablation and Gal-1 inhibition resulted in a significant tumor reduction compared to monotherapy ablation or Gal-1 inhibition alone.

IV. METHODS

Retrospective clinical study

This retrospective study was performed under written-informed consents and approval from UCLA Institutional Board Review (IRB#23–000131). A total of 58 patients who had been diagnosed with moderate-differentiated (n=54) or poorly-differentiated (n=4) HCC, were included in this study. These patient cohorts were matched for demographic, clinical baseline and other tumor characteristics as shown in Table S1.

Mouse Studies

All mouse studies were performed in accordance with UCLA institutional ethical guidelines and detailed in Supporting Methods. Briefly, 5-week-old nude mice (homozygous for Foxn1<nu>, male, cat#002019, Jackson Laboratories) were orthotopically implanted with SNU449-cell-derived tumors. The study subjects were then subject to thermal ablation and/or 5mg/kg OTX008 adminstration with the timeline (Figure 8A).

Mass Spectrometry Data Analyses

Proteomic Analysis

MSFragger software with specific protein sequence database was used for identification. Raw MS1 was calibrated for m/z (mass-to-charge) based on all identified ions. Sample intensities were normalized using sample preparation and injection volumes.

Metabolomic Analysis

Following data acquisition as detailed in Supporting Methods, data were analyzed using Maven (version 8.1.27.11). Metabolites were identified based on accurate mass (±5 ppm) and previously established retention times of pure standards. Data analysis was performed using in-house R scripts.

Statistical Analysis

The log-rank test was utilized for tumor progression-free survival rates. Multiple linear regression, Benjamin-Hochberg, and WGCNA were used in proteomics statistical analysis. Chi-square and unpaired-one-tailed Student’s t-test were used for statistical analysis of clinical characteristics and in vitro and in vivo experiments, respectively. P-values less than 0.05 were considered statistically significant.

Additional details about all the experiments in this study can be found in the Supporting Methods and Materials.

V. RESULTS

THERMAL-ABLATION NONRESPONSE CORRELATES WITH GALECTIN-1 OVEREXPRESSION IN FFPE-NEEDLE-BIOPSY SAMPLES AND HCC CELLS IN VITRO

Thermal ablation has become standard of care for early-stage and non-resectable HCCs within BCLC 0-A criteria.² Most patients experience an initial complete response after ablation, but up to 40% of patients will eventually progress (Figure 1A), despite having similar risk profiles.^3,4 While the center of the ablation zone is associated with immediate cell death⁶, the peripheries are associated with sublethal-hyperthermic microenvironment (Figure S1A) which can promote HCC-aggressive phenotypes.⁵ A retrospective study of patients with matched-propensity scores (Table S1), was conducted using a unique cohort of pre-ablation needle biopsies of early-stage HCCs. Patients were categorized as thermal-ablation responders, defined as those who had a complete response by localized mRECIST criteria for up-to-two years, compared to thermal-ablation nonresponders, defined as those who had progressive disease by localized mRECIST criteria within the two years, a time-frame used in prior work.¹⁵ Ablation-responders (n=34) had significantly longer tumor progression-free survival compared to nonresponders (n=24) (57.0±1.6 (median not reached) versus 8.3±0.5 months (median: 13.6 months),p <0.001), respectively (Figure 1B).

Figure 1: Upregulation of Galectin-1 is correlated with nonresponsiveness to thermal-ablation therapy in HCC.

A) (Top) T1-weighted non-contrast MRI of an HCC patient showing a completely ablated HCC tumor (yellow arrow) and adequate margins (orange arrowhead) around the target HCC. (Bottom) Three-month follow-up post-contrast-arterial phase MRI demonstrating rapid new growth (green arrowhead) around the prior ablation zone. B) Kaplan-Meier curve analysis of tumor progression-free survival probability in HCC patients who had stable disease (responders) versus progressive disease (nonresponders) to thermal-ablation therapy. C)The plot displays protein identifications across 55 Formalin-Fixed Paraffin-Embedded (FFPE) liver tumor samples, stratified by treatment response (n=32 responders and 23 nonresponders). Data points represent individual samples, with responders shown in blue and nonresponders in red. The black line indicates the Loess smoothed trend with 95% confidence intervals (gray shading). Above the main plot, two annotation tracks display z-score-normalized values (white to red gradient) for tumor size and alpha-fetoprotein (AFP) levels, winsorized at ±2 standard deviations. D) Heatmap of differentially expressed proteins (nominal p < 0.05) in tumors classified as responders or nonresponders to ablation. Protein expression values are represented as winsorized Z-scores ranging from −2 to 2, with red indicating higher expression and blue indicating lower expression. Columns correspond to individual samples, clustered by hierarchical clustering, while rows correspond to proteins, sorted first by manually assigned functional category (left color bar) and then by t-value. Sample-associated annotations are displayed at the top, including tumor size (<3 cm vs. >3 cm), AFP levels (<200 ng/mL vs. >200 ng/mL), and response classification (responder vs. nonresponder). Functional categories include cytoskeleton, expression-related, immune-related, metabolism, mitochondria, and trafficking. E) Visualized as a barplot of −log₁₀(P-value) for enriched biological pathways. Pathways enriched in nonresponders (positive enrichment) are shown in red bars extending to the right, while pathways enriched in responders (negative enrichment) are shown in dark blue bars extending to the left. The x-axis represents the −log₁₀(P-value), indicating the statistical significance of enrichment, with higher absolute values reflecting greater significance. F) Upregulated protein levels of Galectin-1. The abundance levels were log₁₀-transformed. Box plots show median (central line), upper and lower quartiles (box limits), and 1.5 interquartile range (whiskers). G) Matched FFPE biopsy samples (n=17 responders and 11 nonresponders) and randomly-selected healthy liver samples (n=11) were stained for Galectin-1 using immunohistochemistry. H) Cells positive for the antibody were quantified using HALO software to calculate mean number of positive cells. The cell numbers were then normalized by total tissue areas. P-value was calculated using one-tailed and unpaired Student’s t-test with *p<0.05, **p<0.01, ***p<0.001.

Proteomic analysis was performed using an untargeted-proteomic approach and identified 1,712 proteins from 32 responsive and 23 nonresponsive FFPE-biopsy samples (Figure 1C). There were 59 proteins that were differentially expressed (nominal p< 0.05) (Figure 1D) and 19 pathways that were found enriched with log₁₀-transformation of significant p-values as shown (Figure 1E). Among the identified proteins, Galectin-1 (Gal-1) was selected for further study due to its previously established association with cancer aggressiveness and treatment resistance,^9–11 and, importantly, because its direct role in HCC metabolism remains largely unexplored. Therefore, understanding the role of Gal-1 in metabolic reprogramming could potentially identify breakthrough and drug-targetable points for improving HCC clinical outcomes. Additionally, Gal-1 levels were analyzed using immunohistochemistry staining on respective samples to further confirm its differential-expression levels found on FFPE proteomic analysis. This analysis revealed that Gal-1 was significantly more expressed in nonresponders compared to responders (Figure 1G-H). Normal liver tissues were also stained for Gal-1, which showed significantly less Gal-1 expression compared with nonresponders but similar to responders (Figure 1G-H).

Next, an in vitro model was created to simulate the clinical thermal-ablation model to further investigate Gal-1 functions. HCC-derived cell lines (SNU423, SNU449, and HepG2/C3a) were exposed to either normothermic (37°C) or sublethal-hyperthermic (47°C) temperatures in a water bath for 25 minutes with the initial 15 minutes for equilibration (Figure S1B-C). Consistent with prior studies simulating the peri-ablational zone,^16,17 sublethal temperature defined as 47°C, was used to induce partial cell death without necrosis to allow for in-depth metabolic investigations. Cell-death percentage under hyperthermia was then assessed at 24, 48, and 72 hours. SNU449 was found to be significantly more resistant against hyperthermia, with a markedly-lower cell-death percentage at 47°C compared to SNU423 (Figure 2A-B). This resistance pattern was verified with bright-field microscopy (Figure S1D), confirming SNU423 and SNU449 as hyperthermia-responsive and resistant cells, respectively. The relation of Gal-1 to hyperthermia resistance, previously noted in FFPE samples (Figure 1F), was then evaluated. Immunoblotting confirmed Gal-1 upregulation in hyperthermia-resistant SNU449 compared to hyperthermia-responsive SNU423 cells (Figure 2C). These findings suggest a link between Gal-1 and thermal-ablation responsiveness in HCC patients and cell lines.

Figure 2: Galectin-1 overexpression induces sublethal hyperthermia resistance in HCC cells.

A-B) SNU449 cell survival percents (n=3 each condition) (A) post thermal exposure at 37°C and 47°C at 24, 48, and 72 hours. Corresponding cell growth curve (B) at the same time intervals. C) Western blot showing the expression of Gal-1 in SNU449, SNU423, and HepG2/C3a (positive control). β-Tubulin expression was used as loading controls. D-E) SNU449 cell-growth curve (n=3 each condition) (D) post-thermal exposure at 37°C and 47°C with Gal-1 selective inhibitor OTX (50μM) or DMSO at 24, 48, and 72 hours. Corresponding cell-survival percentage (E) at the same time intervals. F-G) shControl and shGal-1-SNU449 cell growth curve (n=3 each condition) (F) post-thermal exposure at 37°C and 47°C at 24, 48, and 72 hours. Corresponding cell-survival percentage (G) at the same time intervals. H-I) shGal-1-SNU449 (Gal-1 knockdown, control group) and pGal-1-SNU449 (Gal-1 overexpression using pLentivirus-ORF particles, experimental group) cell growth curve (n=3 each condition) (H) post-thermal exposure at 37°C and 47°C at 24, 48, and 72 hours. Corresponding cell-survival percentage (I) at the same time intervals. P-values were calculated using one-tailed, unpaired Student’s t-test, *p<0.05, **p<0.01, ***p<0.001.

GALECTIN-1 OVEREXPRESSION INDUCES SUBLETHAL-HYPERTHERMIA RESISTANCE IN HCC CELLS IN VITRO

Given the observed Gal-1 overexpression in hyperthermia-resistant SNU449 (Figure 2C), selective modulation of Gal-1 was performed to isolate its role in promoting hyperthermia resistance. SNU449 was treated with a pre-determined dose of 50μM Gal-1 inhibitor (OTX008) (Figure S2A-B) before thermal exposure.¹¹ SNU449 growth in the OTX-treated group under hyperthermia was markedly diminished compared to those only exposed to hyperthermia at 47°C (Figure 2D). Cell-death percentage demonstrated that Gal-1 inhibition also significantly increased hyperthermic sensitivity (Figure 2E). Similar increases in hyperthermic sensitivity were noted in SNU423 with Gal-1 inhibition (Figure S2C-D).

Gal-1 was then silenced using lentiviral-shRNA particles in SNU449 (shGal-1-SNU449) (Figure S3A). Growth-rate analysis showed shGal-1-SNU449 was significantly more susceptible to hyperthermia than shControl-SNU449 (Figure 2F). Cell-survival studies corroborated the cell-growth results (Figure 2G), further suggesting that silencing Gal-1 enhanced hyperthermic sensitivity. To investigate the rescuing effect of increased Gal-1 expression, Gal-1-knockdown SNU449 (shGal-1-SNU449) was then overexpressed with Gal-1 using pLentivirus-ORF particles (pGal-1) (Figure S3B). Cell-growth and survival analyses under hyperthermia showed that pGal-1 was significantly more resistant to hyperthermia than its respective-control shGal-1 (Figure 2H-I). Altogether, these findings underscore the critical role of Gal-1 in promoting hyperthermia resistance in HCC.

GALECTIN-1 OVEREXPRESSION INCREASES THE COMPLEX FORMATION BETWEEN GALECTIN-1 AND N-TERMINAL OF P-GLYCOPROTEIN VIA AN O-GLCNACYLATION-DEPENDENT PATHWAY UNDER HYPERTHERMIA IN HCC CELLS

To further understand the role of Gal-1 in hyperthermia resistance, Gal-1 expression was assessed under hyperthermia and normothermia in shControl-SNU449 and shGal-1-SNU449. This analysis revealed no significant changes in Gal-1 (15 kDa) levels in both cell lines (Figure 3A). Because Gal-1 is co-localizing with GM1 (monosialotetrahexosyl-ganglioside),¹⁸ a GM1-immunoblotting study was performed by using cholera-toxin subunit B (CTBx) and CTBx antibody.¹⁹ This analysis showed a significant upregulation of a protein around 105 kDa after hyperthermia exposure in both cell lines (Figure 3B). This protein was investigated further and found to be composed of Gal-1 because the same 105 kDa band appeared when Gal-1 was probed (Figure 3C). In addition to Gal-1 and GM1, the protein makeup was suspected to require additional components because Gal-1 and GM1 together still did not fully account for the total weight. Prior work has suggested a strong affinity between P-glycoprotein (P-gp), an ATP-binding transporter, and Gal-1.^18,20 Specifically, Gal-1 binds to N-terminal of P-gp (N-P-gp) in the cytosol.^18,21 Therefore, it was hypothesized that reducing P-gp, and consequently N-P-gp, would decrease the 105 kDa-protein expression.

Figure 3: Galectin-1 overexpression increases the complex formation between Gal-1 and N-terminal of P-glycoprotein via an O-GlcNAcylation-dependent pathway under hyperthermia (47°C) in HCC cells.

A) Western blot showing levels of Galectin-1 (Gal-1) in shGal-1-SNU449 (SNU449 cells with Gal-1 silencing via lentiviral particles) and respective shControl-SNU449 cells after thermal exposure at 37°C or 47°C. β-Tubulin expression was used as loading controls. N=2 duplicates per group. B-C) Western blot showing levels of GM1 binding to the complex formed by N-terminal-half of P-glycoprotein and Gal-1 (N-P-gp/Gal-1) in shGal-1-SNU449 and respective shControl-SNU449 cells after thermal exposure at 37°C or 47°C. GM1 levels were measured using western blot to stain for cholera toxin B (CTBx) subunits which have high affinity for GM1. Therefore, after the thermal treatments, cells were added 10μM of CTBx to stain for GM1. The cells were then harvested and stained using polyclonal CTBx antibody for GM1 staining (B) or Gal-1 for Gal-1 staining (C). N=2 duplicates per group. Dotted lines indicate discontinuous wells on the same membrane. D-E) Western blot showing levels of GM1 binding to the complex formed by N-terminal-half of P-glycoprotein and Gal-1 (N-P-gp/Gal-1), in SNU449 WT treated with DMSO or Wortmannin (Wort, 200μM) to reduce P-gp, and subsequently N-terminal of P-gp expression. Immunoblotting was performed using polyclonal CTBx antibody for GM1 staining (D) or Gal-1 for Gal-1 staining (E). N=2 duplicates per group. Dotted lines indicate discontinuous wells on the same membrane. F) Western blot showing O-GlcNAcylation (O-GlcNAc) levels post-thermal exposure. PUGNAc (100μM) was used to treat to preserve the full production of O-GlcNAc in shControl-SNU449 cells 24 hours prior to thermal exposure. Immunoblotting was then performed using O-GlcNAc antibody. G) shControl-SNU449 cells were treated with DON (L-6-Diazo-5-oxonorleucine) (400μM) to inhibit O-GlcNAc for 3 days before thermal exposure. Cytoplasmic fractions were extracted using NuCLEAR extraction kit. Immunoblotting was performed using Gal-1 antibody. α-tubulin was used as loading controls. H) Illustration showing the mechanistic association between thermal stress and formation of GM1/Gal-1/N-P-gp complex. I) After DON and thermal treatments as shown in G), shControl-SNU449 cells were added 10μM of CTBx to stain for GM1. Immunoblotting was then performed using polyclonal CTBx antibody for GM1 staining. J) After DON and thermal treatments as shown in G), shControl-SNU449 and shGal-1-SNU449 cells were added 10μM of CTBx to stain for GM1. Immunoblotting was then performed using polyclonal CTBx antibody for GM1 staining. Dotted lines indicate discontinuous wells on the same membrane. K) Illustration showing the correlation between Gal-1 expression and formation of GM1/Gal-1/N-P-gp complex under hyperthermia.

To reduce P-gp, a selective inhibitor (Wortmannin/Wort) of PI3K pathway regulating P-gp expression,¹⁰ was used under normothermia at a pre-determined concentration from a titration study (Figure S4A). This investigation was performed in SNU449-WT (wild type) as these cells were overexpressed with P-gp (Figure S4B). GM1 (Figure 3D) and Gal-1 (Figure 3E) were then probed, and both demonstrated a decrease in the 105 kDa-protein expression upon P-gp reduction. These findings collectively indicate that the 105 kDa protein was formed by GM1 binding to Gal-1, which then binds to N-terminal of P-gp.

The mechanism of hyperthermia-induced upregulation of GM1/Gal-1/N-P-gp complex (Figure 3B-C) was then evaluated. P-gp binds to Gal-1 via N-acetylglucosamine residues on P-gp.^18,20,21 These residues are subject to O-GlcNAcylation (O-GlcNAc), a stress-responsive post-translational modification that mediates the binding interactions between O-linked N-acetylglucosamine with surrounding proteins.²² Furthermore, O-GlcNAc has been found to be modified by hyperthermia exposure in HCC.^16,22 Therefore, we hypothesized that O-GlcNAc may impact the formation of GM1/Gal-1/N-P-gp complex under hyperthermia. shControl-SNU449 was used for this study because its Gal-1 overexpression would allow for the opportunity to analyze the formation of GM1/Gal-1/N-P-gp. First, O-GlcNAc was assessed and found to be significantly decreased in shControl-SNU449 under hyperthermia compared to normothermia (Figure 3F). PUGNAc (100μM), an O-GlcNAc-breakdown inhibitor,¹⁶ was used to preserve the full O-GlcNAc production, allowing for a complete evaluation of the hyperthermic impact. The relationship between Gal-1 and O-GlcNAc reduction under hyperthermia was then investigated. Specifically, cytosolic Gal-1 levels were assessed because O-GlcNAc occurs in the cytosol.²² When O-GlcNAc was maximally suppressed by combining hyperthermia with O-GlcNAc inhibition (L-6-Diazo-5-oxonorleucine, (DON)),²³ cytosolic Gal-1 was found to be significantly reduced compared to either hyperthermia or DON-treated alone groups (Figure 3G). This finding indicates that decreased O-GlcNAc led to increased Gal-1 secretion and consequently reduced Gal-1 remaining in cytosol, consistent with prior work.²³ As the number of Gal-1 molecules translocating through the membrane increased upon O-GlcNAc reduction, the interactions between Gal-1 and a membrane protein like P-gp would increase.²⁴ Thus, the formation of GM1/Gal-1/N-P-gp would hypothetically increase upon decreased O-GlcNAc levels under hyperthermia as illustrated (Figure 3H). Expectedly, when O-GlcNAc level was maximally suppressed in the combined hyperthermia/DON-treated group, GM1/Gal-1/N-P-gp complex exhibited the largest increase in expression compared to either hyperthermia or DON-treated alone (Figure 3I). In addition to being mediated by O-GlcNAc, GM1/Gal-1/N-P-gp complex formation was demonstrated to be dependent on Gal-1 expression. When O-GlcNAc was suppressed maximally in the combined hyperthermia/DON-treated group, GM1/Gal-1/N-P-gp was significantly more expressed in Gal-1-overexpressing-shControl-SNU449 than in Gal-1-underexpressing-shGal-1-SNU449 (Figure 3J-K). Overall, these findings demonstrate that the formation of GM1/Gal-1/N-P-gp complex under hyperthermia depends on both O-GlcNAc and Gal-1 expression.

GALECTIN-1 COMPLEXES WITH THE N-TERMINAL OF P-GLYCOPROTEIN TO MODULATE GLYCOLYSIS VIA GM1-HYDROLYZED GALACTOSE PRODUCTION

The signaling functions of Gal-1 have been extensively studied,^10,11 but its direct role in HCC metabolism remains unclear. Glycolysis, a key metabolic pathway in HCC and many other cancers, is crucial for tumor progression.¹⁴ The FFPE-biopsy-proteomic analysis revealed enrichment of glycolysis-related pathways (Figure 1E), including β-catenin-mediated glycolysis via pyruvate dehydrogenase kinase isozyme 1 expression and NOTCH-mediated glycolysis via p53 or PI3K/AKT signaling.²⁵ Furthermore, levels of glycolysis-related proteins (Figure 4A)—glucose 6-phosphate isomerase (GPI), glyceraldehyde-3-phosphate dehydrogenase (GADPH), phosphoglycerate kinase (PGK)—from the differential-expression-protein analysis (Figure 1D), were also found to be upregulated in nonresponders compared to responders (Figure 4B-D). These findings led us to specifically characterize the mechanistic underpinnings between increased glycolysis and Gal-1 overexpression. This investigation started with SNU423 as hyperthermia-responsive and SNU449 as hyperthermia-resistant cells, respectively (Figure 2A-B). Glycolysis was noted to be significantly upregulated in SNU449 compared to SNU423, resulting in increased glycolytic ATP production (Figure 4E-F). When Gal-1 was inhibited with OTX in SNU449 WT, glycolysis was expectedly decreased along with a concomitant decrease in glycolytic ATP production (Figure 4G-H), establishing the direct link between Gal-1 and glycolysis.

Figure 4: Galectin-1 overexpression is associated with glycolytic resistance via a GM1-dependent pathway.

A) Illustration showing glycolysis pathway and associated metabolites and catalytic enzymes. B-D) Upregulation of glycolytic proteins, Glucose-6-Phosphase Isomerase (GPI) (B), Glyceraldehyde 3-Phosphate Dehydrogenase (GADPH) (C), Phosphoglycerate Kinase (PGK) (D) in HCC nonresponders (n=23) compared to responders (n=32). The abundance levels were log₁₀-transformed. Box plots show median (central line), upper and lower quartiles (box limits), and 1.5 interquartile range (whiskers). E) Glycolytic activities in SNU449 and SNU423 (n=5 each condition) were measured based on the basal extracellular acidification rates (ECAR) via a Seahorse XF96 analyzer. F) ATP production rates via glycolysis in SNU449 and SNU423 were measured using a Seahorse XF96 analyzer. G) Glycolytic activities in SNU449 with Gal-1 inhibitor OTX (150μM) or DMSO (vehicle control) (n=10 each condition) were measured. H) Corresponding ATP production rates under the same conditions. I) Schematic diagram showing the interactions between Galectin-1 and β-Galactosidase with P-gp as well as between β-Gal and GM1 ganglioside. J) Western blot showing levels of β-Gal binding to N-P-gp proteins after SNU449 WT cell were treated with Wort (200μM) to reduce N-P-gp expression. β-Tubulin expression was used as loading controls. N=2 duplicates per group. Dotted lines indicate discontinuous wells on the same membrane. K) Western blot showing levels of β-Gal binding to N-P-gp proteins in SNU449 WT treated with PBS or Caffeine (β-Gal inhibitor) (15mM). N=2 duplicates per group. Dotted lines indicate discontinuous wells on the same membrane. L) Western blot showing levels of GM1 binding to the complex formed by N-P-gp and Gal-1 in the corresponding conditions as shown in (K). N=2 duplicates per group. Dotted lines indicate discontinuous wells on the same membrane. P-value was calculated using one-tailed and unpaired Student’s t-test with **p<0.01, ***p<0.001.

The specific mechanism of Gal-1-mediated glycolysis was then evaluated. Increase in the complex formation of GM1/Gal-1/N-P-gp was identified under hyperthermia (Figure 3B-C), providing the basis for our rationale. β-galactosidase (β-gal) then emerged as a key molecule of interest due to its role in catalyzing GM1 hydrolysis to produce galactose,²⁶ a precursor for the glycolytic substrate glucose 6-phosphate. β-gal has a high affinity for the N-terminal of P-gp which separately binds to Gal-1 as illustrated (Figure 4I).^21,26,27 Therefore, P-gp expression reduction, and consequently N-P-gp expression, would reduce N-P-gp/β-gal complex. To test this, P-gp expression was reduced using Wort, leading to a decrease in N-P-gp/β-gal at approximately 193 kDa (Figure 4J), consistent with prior work.²⁷ To confirm the association of N-P-gp and β-gal, a selective β-gal inhibitor (Figure S4C), was used. Expectedly, N-P-gp/β-gal complex levels were increased upon β-gal inhibition (Figure 4K). This trend suggests that reduced β-gal catalytic activity decreased its dissociation from N-P-gp. Consequently, β-gal inhibition decreased GM1 hydrolysis, resulting in increased intact GM1 to bind Gal-1 that binds to N-P-gp (Figure 4L). Collectively, these findings further support the mechanism of Gal-1-mediated glycolysis via a N-P-gp/β-gal-GM1-dependent pathway as illustrated (Figure 4I).

The proposed mechanism (Figure 5A) indicates that Gal-1 overexpression would allow cells to hydrolyze more GM1 to produce more galactose. To further study this, galactose levels were then measured in Gal-1-overexpressing-shControl and Gal-1-underexpressing-shGal-1 using an Abcam-Galactose assay. This analysis showed a significant decrease in galactose in shGal-1 compared to shControl (Figure 5B). Moreover, the catalytic activity of β-gal in GM1 hydrolysis to produce galactose was reduced in shGal-1 compared to shControl (Figure 5C). To confirm if decreased galactose would reduce glycolysis, ECAR (extracellular acidification rates, an indicator of glycolysis) was assessed. Compared to shControl, shGal-1 showed a marked reduction in glycolysis and glycolytic ATP production (Figure 5D-E). These findings suggest that galactose metabolism, and thus glycolytic influx, is less upregulated in shGal-1.

Figure 5: Hydrolysis of Galectin-1-bound GM1 mediates glycolysis through galactose production.

A) Schematic diagram showing the mode of action of Gal-1 binding to GM1 and P-gp to bridge GM1 and its catalytic enzyme, β-Gal. These interactions ultimately lead to GM1 breakdown to produce galactose and subsequent glucose-6-phosphate for glycolysis. B) Galactose levels were assessed in shControl and shGal-1 (n=3 each condition) using an Abcam galactose assay kit. RFU = relative fluorescence units. C) β-Galactosidase (β-Gal) activity was assessed in shControl and shGal-1-SNU449 (n=3 each condition). D) Glycolytic activities in shControl and shGal-1 (n=5 each condition) were measured based on the basal extracellular acidification rates (ECAR) via a Seahorse XF96 analyzer. E) ATP production rates via glycolysis in shControl and shGal-1 were measured using a Seahorse XF96 analyzer. F) Schematic diagram showing the metabolism of galactose via Leloir pathway and subsequently glycolysis. This diagram also shows the isotopologue tracing when U-¹³C₆ galactose is used. G) Percent enrichment of galactose-1 phosphate (Gal-1P) product from the metabolism of U-¹³C₆ galactose in shControl and shGal-1-SNU449 cells (n=3 each condition). H) Percent enrichment of remaining U-¹³C₆ galactose after its metabolism in shControl and shGal-1-SNU449 cells. I-K) Percent enrichment of glucose-6 phosphate (G6P) (M+6) (I), 3/2 phosphoglycerate (M+3) (J), lactate (M+3) (K) products from the metabolism of U-¹³C₆ galactose in shControl and shGal-1-SNU449 cells (n=3 each condition). P-values were calculated using one-tailed, unpaired Student’s t-test, *p<0.05, **p<0.01, ***p<0.001.

To validate this implication, U-¹³C₆ galactose, a carbon-13-labeled form of D-Galactose, was utilized to trace galactose metabolism within the Leloir pathway, the primary metabolic pathway for galactose utilization,²⁸ and glycolysis (Figure 5F). U-¹³C₆ galactose-1-phosphate (Gal-1P, M+6) was increased in shGal-1 (Figure 5G), suggesting that shGal-1 cells were not as heavily consuming Gal-1P to promote increased glycolysis as shControl cells. Additionally, a significantly higher concentration of unmetabolized U-¹³C₆ galactose was found in shGal-1 (Figure 5H), further confirming the downregulation of Leloir pathway in shGal-1. The products of U-¹³C₆ galactose metabolism were then traced for their contribution to downstream glycolysis. Consistent with decreased glycolysis in Gal-1-underexpressing-shGal-1 cells (Figure 5D), glycolytic metabolite U-¹³C₆ glucose-6 phosphate (G6P, M+6) was decreased in shGal-1 compared to shControl (Figure 5I). Other downstream glycolytic metabolites, 3/2-phosphoglycerate (3/2-PG, M+3) (Figure 5J) and lactate (Lac, M+3) (Figure 5K), were also markedly reduced in shGal-1 compared to shControl. In summary, these findings confirm the role of Gal-1-mediated glycolysis via galactose production to support HCC proliferation.

GALECTIN-1 SILENCING AND INHIBITION FURTHER REDUCE METABOLITES AND ACTIVITIES OF GLYCOLYSIS AND MITOCHONDRIAL TCA CYCLE UNDER HYPERTHERMIA

Thus far, the present study has demonstrated the mechanism by which Gal-1 mediates glycolysis in HCC under normothermia (Figure 5A). We then sought to determine the effects of Gal-1 modulation by genetic silencing or pharmacological inhibition under hyperthermia by examining the metabolites and activities of glycolysis and downstream TCA cycle (tricarboxylic acid). This study employed U-¹³C₆ glucose (M+6), a carbon-13-labeled form of D-Glucose, to track glucose metabolism in Gal-1-overexpressing-shControl and Gal-1-underexpressing-shGal-1 (Figure 6A). Lactate, a glycolysis-end product whose concentration reflects glycolytic activity,¹⁴ (M+3)-isotopologue levels were significantly reduced in shGal-1 compared to shControl under hyperthermia (Figure 6B). Moreover, glycolytic metabolite phosphoglycerate (3/2-PG) (M+3) was reduced in shGal-1 compared to shControl under hyperthermia (Figure 6C). These findings suggest low Gal-1 expression further reduces glycolysis under hyperthermia. Additionally, metabolites of TCA cycle were further decreased in shGal-1 compared to shControl under hyperthermia. Specifically, citrate (M+0 to M+6) isotopologues were markedly reduced in shGal-1 compared to shControl under hyperthermia (Figure 6D). Similarly, malate (M+0 to M+4) levels were significantly decreased in shGal-1 compared to shControl under hyperthermia (Figure 6E). Total levels of lactate (Figure 6F), 3/2-PG (Figure 6G), citrate (Figure 6H), and malate (Figure 6I) were also measured, which consistently showed significant decreases in shGal-1 compared to shControl under hyperthermia. These metabolic reductions could link decreased Gal-1 expression with significant reductions in glycolysis and TCA cycle activities. To confirm this implication, the glycolytic and mitochondrial TCA cycle activities were measured under the hyperthermic conditions by using a Seahorse analyzer to quantify ECAR (an indicator of aerobic glycolysis) and OCR (oxygen consumption rate, an indicator of TCA cycle activity via oxidative phosphorylation).²⁹ This analysis showed marked decreases in glycolysis and TCA cycle activities (Figure 6J-K) along with reduced ATP production rates (Figure S5A-B) in shGal-1 compared to shControl under hyperthermia.

Figure 6: Galectin-1 silencing reduces metabolites and activities of glycolysis and mitochondrial TCA cycle under hyperthermia.

A) Schematic diagram showing the isotopologue tracing of U-¹³C₆ glucose metabolism via glycolysis and TCA cycle. B-E) shControl and shGal-1-SNU449 cells (n=3) were cultured with U-¹³C₆ glucose (10mM) in glucose-free RPMI and then exposed to 37°C or 47°C.The metabolic extraction was performed 3 hours post thermal exposures. The metabolic abundances were then assessed using mass spectrometry. These levels were then compared to the respective controls to evaluate the relative changes in the enrichments among the experimental conditions. The isotopologues (lactate (M+3) (B), 3- or 2-phosphoglycerate (3/2-PG) (M+3) (C),citrate (M+2 to M+6) (D), malate (M+0 to M+4) (E)) presented here were those that directly originated from U-¹³C₆ glucose or its direct intermediates. F-I) the total abundances of lactate (E), 3/2-PG (G), citrate (H), and malate (I) were also quantified. These levels were then compared to their respective controls to evaluate metabolic changes among the experimental groups. J-K) Glycolytic (J) and TCA cycle (K) activities in shControl and shGal-1 under hyperthermia (47°C) and normothermia (37°C) (n=10 each condition) were measured based on the basal extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) via a Seahorse XF96 analyzer. P-values were calculated using one-tailed, unpaired Student’s t-test, *p<0.05, **p<0.01, ***p<0.001.

To further investigate the effects of Gal-1 modulation on glycolysis and TCA cycle, the isotopologue tracings of U-¹³C₆ glucose metabolism was performed in SNU449 WT with Gal-1 inhibition (OTX) (Figure 7A). Compared to control, the Gal-1-inhibited group exhibited reductions in various glycolytic metabolites, lactate (M+3), fructose 1,6-bisphosphate (F1,6BP) (M+6), 3/2-PG (M+3), and phosphoenolpyruvate (PEP) (M+3), under hyperthermia (Figure 7B-E). Also, the Gal-1-inhibited group exhibited significant decreases in citrate (M+0 to M+6) and malate (M+0 to M+4) within the TCA cycle compared to the control under hyperthermia (Figure 7F-G). Total levels of these metabolites were then measured and showed significant reductions in the Gal-1-inhibited group compared to the control under hyperthermia (Figure 7H-M). To confirm whether the metabolic decline would lead to reduction in the activities of glycolysis and downstream TCA cycle, ECAR and OCR, respectively, were quantified. Indeed, this analysis showed significant decreases in the activities of glycolysis and TCA cycle (Figure 7N-O) as well as associated ATP production rates (Figure S5C-D) in the Gal-1-inhibited group compared to control under hyperthermia. Collectively, these findings underscore the importance of Gal-1 in facilitating energy production via glycolysis and TCA cycle for HCC resistance against hyperthermia-induced stress.

Figure 7: Galectin-1 inhibition reduces metabolites and activities of glycolysis and mitochondrial TCA cycle under hyperthermia.

A) Schematic diagram showing the isotopologue tracing of U-¹³C₆ glucose metabolism with Gal-1 inhibition (OTX) via glycolysis and TCA cycle. B-E) SNU449 WT cells (n=3) were cultured with U-¹³C₆ glucose (10mM) in glucose-free RPM and treated with OTX (150μM) or DMSO (vehicle control) for 24 hours before exposing cells to 37°C or 47°C.The metabolic extraction was performed 3 hours post thermal exposures. The metabolic abundances were then assessed using mass spectrometry. These levels were then compared to the respective controls to evaluate the relative changes in the enrichments among the experimental conditions. The isotopologues, lactate (M+3) (B), fructose 1,6bisphosphate (F1,6BP, M+6) (C), 3- or 2-phosphoglycerate (3/2-PG) (M+3) (D), phosphoenolpyruvate (PEP, M+3) (E), citrate (M+0 to M+6) (F), malate (M+0 to M+4) (G)) presented here were those that directly originated from U-¹³C₆ glucose or its direct intermediates. H-M) the total abundances of lactate (H), F1,6BP (I), 3/2-PG (J), PEP (K), citrate (L), and malate (M) were also quantified. These levels were then compared to their respective controls to evaluate metabolic changes among the experimental groups. N-O) Glycolytic (N) and TCA cycle (O) activities in SNU449 WT with Gal-1 inhibition (150μM OTX) under hyperthermia (47°C) and normothermia (37°C) (n=10 each condition) were measured based on the basal extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) via a Seahorse XF96 analyzer. P-values were calculated using one-tailed, unpaired Student’s t-test, *p<0.05, **p<0.01, ***p<0.001.

TARGETING GALECTIN-1 BY SELECTIVE INHIBITOR OTX008 ENHANCES THERMAL-ABLATION EFFICACY IN HYPERTHERMIA-RESISTANT-HCC TUMORS

Hyperthermia-resistant SNU449 cells were used to evaluate the efficacy of Gal-1 inhibition in improving post-ablation tumor control an orthotopic model. This cell line was used because success in controlling its tumor progression would further emphasize the critical role of the Gal-1 in mediating glycolysis and consequently the TCA cycle in promoting metabolic plasticity. After subcutaneous SNU449-cell-derived tumors were orthotopically implanted into the livers of 5-week-old-male-nude mice, each subject was administered with OTX prior to ablation as shown (Figure 8A). In accordance with institutional ethical guidelines, mice were sacrificed one-week post-ablation, and tumors were harvested. Tumor growth was significantly reduced in the combined ablation and OTX-treated group compared to ablation or OTX alone (Figure 8B-C). There was no tumor reduction observed in the monotherapy ablation compared to control, which was expected because SNU449 cells are resistant to hyperthermia (Figure 2A-B, S1C-D).

Figure 8: Targeting Galectin-1 by selective inhibitor OTX008 enhances thermal-ablation efficacy in hyperthermia-resistant-HCC tumors.

A) Schematic diagram showing the timeline for tumor implantation and for drug OTX and thermal ablation treatments in vivo. B) Images of tumors from the 4 study groups: S-ablation DMSO (S=sham) (n=6), ablation DMSO (n=6), S-ablation OTX (n=6), ablation OTX (n=5, O = complete disappearance). C) Tumor weights from the 4 study groups (n=6 for S-ablation DMSO, Ablation DMSO, and S-Ablation OTX, n=4 for Ablation OTX). D) Galactose levels were assessed in the tumors from the four study groups, by using an Abcam galactose assay kit. These levels were then compared to the control (S-ablation DMSO) to evaluate for galactose changes among the study groups. E) Schematic diagram showing galactose metabolism (Leloir pathway) and glycolysis. F) 50mg of the harvested tumors from all groups were subject to metabolic extraction and submitted to mass spectrometry to assess levels of glucose-1 phosphate (Gal-1P). G) Western blot showing expression levels of Gal-1 binding to N-terminal-half of P-glycoprotein (N-P-gp/Gal-1) and free Gal-1 in the tumors from the four study groups. β-Tubulin expression was used to as loading controls. N=2 duplicates per group. H-K) Metabolic extracts from the tumors were also assessed for fructose-1,6-bisphosphate (F1,6BP) (H), phosphoenolpyruvate (PEP) (I), citrate (Cit) (J), and malate (Mal) (K). These levels were then compared to the control to evaluate for metabolic changes among the study groups. P-values were calculated using one-tailed, unpaired Student’s t-test, *p<0.05, **p<0.01, ***p<0.001.

The tumor-metabolic profile was then characterized using an Abcam-Galactose assay. Galactose levels were substantially reduced in the combined-treatment group compared to thermal-ablation or OTX alone (Figure 8D). Levels of galactose-metabolism product, Gal-1P (Figure 8E), were also measured using mass-spectrometry, which showed a marked decrease in the combined-treatment compared to ablation or OTX alone (Figure 8F). These findings suggest reduced galactose metabolism in the combined-treatment group, potentially due to decreased Gal-1 expression. This aligns with previous in vitro studies linking decreased galactose metabolism to reduced Gal-1 (Figure 5B). To further confirm this, tumor Gal-1 levels were assessed, which showed the largest Gal-1 decrease in the combined-treatment group (Figure 8G). While unbound-Gal-1 expression was decreased, the levels of Gal-1 binding to N-P-gp were increased in the combined-treatment or thermal-ablation groups compared to control (Figure 8G). This finding aligns with our previous observation of increased Gal-1/N-P-gp complex under hyperthermia (Figure 3C). Glycolytic metabolites, F1,6BP and PEP, (Figure 8E) were assessed and found to be markedly decreased in the combined-treatment compared to ablation or OTX alone groups (Figure 8H-I), further confirming decreased glycolysis. Citrate and malate, TCA cycle metabolites, (Figure 8J-K) were also markedly reduced in the combined group compared to ablation or OTX alone, corroborating previous in vitro findings of decreased TCA cycle metabolites upon Gal-1 inhibition (Figure 7L-M). Collectively, the findings in vitro and in vivo highlight the critical role of Gal-1 in mediating glycolysis and consequently the downstream TCA cycle to allow cells to meet their increased-energy demand under hyperthermia.

VI. DISCUSSION

This study investigated the role of Gal-1 in mediating glycolysis and consequently the downstream TCA cycle via a GM1-galactose-dependent pathway to promote hyperthermia resistance and post-ablation progression in early-stage and non-resectable HCC. Gal-1 upregulation was retrospectively identified from the pre-ablation FFPE biopsy samples of thermal-ablation nonresponders. Gal-1-overexpression was then linked to HCC tumor cells’ enhanced ability to utilize glycolysis and downstream TCA cycle under sublethal hyperthermia-induced stress. This process was facilitated by upregulating the formation of GM1/Gal-1/N-P-gp complex via an O-GlcNAcylation-dependent pathway, to bridge GM1 to the catalytic-enzyme β-gal. Additionally, Gal-1 inhibition (OTX008) or knockdown, sensitized HCC cells to sublethal hyperthermia by diminishing the metabolic fluxes and activities of glycolysis and downstream TCA cycle. Importantly, in vivo studies using an orthotopic murine model showed that the combination of Gal-1 inhibition and ablation led to significant tumor-size reduction compared to ablation alone. These results suggest that Gal-1 mediation of glycolysis and consequently the TCA cycle may contribute to thermal-ablation resistance and post-ablation progression in early-stage HCC.

Thermal ablation causes immediate cell death at the center of the ablation zone.^2,6 However, the peri-ablational zones associated with hyperthermia have been correlated with rapid tumor progression in more aggressive subtypes of HCC.^5,16,17 Hyperthermia-induced glycolysis has been proposed to be a key factor contributing to HCC tumor progression.¹⁶ The present study extends beyond glycolysis to reveal the critical role of the TCA cycle in HCC post-ablation progression. These mechanisms of action are found to be specifically orchestrated by Gal-1, a glycan-binding protein whose overexpression correlates with aggressive clinicopathological features such as tumor invasion, angiogenesis cancers, and drug resistance.^9–11 Unlike these prior studies focusing on the genetic-regulatory functions of Gal-1 in cancer aggressiveness, this study demonstrates the direct metabolic regulation of Gal-1 in HCC progression, particularly post-ablation.

Using an in vitro peri-ablational hyperthermia model in the present study, Gal-1 was shown to directly modulate aerobic glycolysis via GM1 hydrolysis to produce galactose. The galactose metabolite is then metabolized to produce glucose 6-phosphate for glycolysis. This glycolytic modulation consequently led to corresponding changes in the metabolic levels and activity of the downstream TCA cycle. Galactose metabolism (Leloir pathway) combined with glycolysis has been hypothesized to yield zero-net ATP compared to glycolysis alone, due to the energy cost of producing UDP-glucose.^30,31 Nevertheless, the produced UDP-glucose can participate in multiple catalytic cycles to generate UDP-galactose, thereby offsetting the initial energy expenditure.³² Ultimately, the combined pathways will still yield two-net ATP molecules and result in increases in the glycolytic influx and consequently the metabolic influx and activity of the TCA cycle for further ATP production.²⁸ Ultimately, this mechanism allows Gal-1-overexpressing HCCs to meet increased-energy demand under hyperthermia. Gal-1 can thus serve as a promising therapeutic target, particularly for HCC patients with an elevated risk of recurrence following ablation.⁷

The in vivo aspect of this study provides key insights into the therapeutic potential of targeting Gal-1 in combination with ablation. This combined strategy led to significantly greater reductions in tumor size and metabolic levels of glycolysis and downstream TCA cycle compared to either ablation or OTX-treatment alone. These reductions also correlated with Gal-1 downregulation in the combined group. Gal-1 downregulation was likely due to increased activity of the 26S proteasome, which degraded the complex formed by Gal-1 and Gal-1 inhibitor OTX at a faster speed under hyperthermia.^11,33 These results indicate that systematically administering Gal-1 inhibitor (OTX008) beforehand to form a Gal-1-OTX complex that is then rapidly degraded under thermal ablation, may provide significant clinical insights. This approach would lead to a reduction in glycolysis and consequently TCA cycle activity, both of which correlate with poor prognosis in HCC patients.^34,35 The safety of Gal-1 inhibitor OTX008 has previously been demonstrated in a first-in-man trial in 2013, where patients with treatment-refractory metastatic colorectal cancer were administered with OTX008 as a daily-subcutaneous injection.³⁶ The dose escalation study showed that a daily-subcutaneous injection at 65mg was safe without dose-limiting toxicity, although plasma concentration was dependent on body weight. Overall, the strategy of combining thermal ablation with a Gal-1 inhibitor holds considerable clinical potential for improving patient outcomes.

Additionally, targeting Gal-1 offers broader clinical implications for improving HCC patient outcomes. As HCC biopsy becomes more widely accepted, biomarkers associated with upregulated aerobic glycolysis and poor survival in HCC (e.g., pyruvate kinase M2 or hexokinase-2)³⁵ can identify patients who would benefit from neoadjuvant-Gal-1-inhibition therapy. Improving local control through Gal-1 modulation can also potentially keep an early-stage-HCC patient within the Milan criteria and reducing the risk for transplant drop-off.² For late-stage HCC, Gal-1 has been established as a potential mediator for angiogenesis and promoter of immune evasion in late-stage HCC^9,37—where checkpoint inhibition and anti-angiogenic drugs have taken center stage.³⁸ Gal-1 overexpression, known to upregulate PD-1/PDL-1 ligands and induce immunotherapy resistance in other tumors,³⁷ suggests that Gal-1 inhibition could enhance checkpoint inhibitor efficacy in HCC. Given the implicated roles of Gal-1 in both early- and late-stage HCC, its inhibition offers a potential adjuvant or neoadjuvant approach to extend response duration across all stages.

There are several limitations in this study. The biopsy samples from early-stage HCCs may be subject to intra-tumoral heterogeneity. However, we focused on evolutionarily-conserved biomarkers across multiple cell types to mitigate the heterogeneity risks. Another limitation of the in vivo studies is that they were performed using a single HCC cell line (SNU449). Nevertheless, this thermally-resistant model was effective in highlighting the potential of Gal-1 inhibition in overcoming ablation-resistance. Future studies will be useful in validating the results in patient-derived HCC tumors. Furthermore, the focus on the roles of Gal-1 in mediating glycolysis and consequently TCA cycle, while important for tumor metabolism, may have also inadvertently overlooked other pathways, namely Gal-1-mediated lipid metabolism.³⁹ Finally, longer follow-up in vivo is required for a comprehensive assessment of survival benefits of Gal-1 inhibition.

Rapid post-ablation progression for early-stage, non-resectable HCC has been an active area of research, particularly because ablation is often applied with curative intent or as a bridge to transplant². This present study provides compelling evidence that Gal-1 plays a critical role in regulating metabolic plasticity in HCC after ablation. By facilitating glycolysis via a GM1-Galactose-dependent pathway and consequently the TCA cycle, Gal-1 enables HCC cells to persist under hyperthermic conditions. Inhibiting Gal-1 can potentially disrupt this metabolic adaptation, which was demonstrated in in vitro and in vivo. Thus, combining Gal-1 inhibitor with ablation can potentially improve patient outcomes post-ablation. While additional research is needed to fully understand the broader metabolic impacts of Gal-1 inhibition, this study represents an important step forward in the development of more effective treatments for early-stage, nonresectable HCC.

List of Abbreviations:

AASLD

American association for the study of liver diseases

ACR

American College of Radiology

AFP

Alpha-fetoprotein

β-gal

Beta-Galactosidase

CTBx

Cholera toxin subunit B

DON

L-6-Diazo-5-oxonorleucine

ECAR

Extracellular acidification rate

FFPE

Formalin-fixed paraffin-embedded

F1,6BP

Fructose 1,6-bisphosphate

Gal-1

Galectin-1

Gal

Galactose

Gal-1P

Galactose 1-phosphate

G6P

Glucose 6-phosphate

GPI

Glucose-6-Phosphase Isomerase

GADPH

Glyceraldehyde 3-Phosphate Dehydrogenase

HCC

Hepatocellular carcinoma

IQR

Interquartile range

Lac

Lactate

LI-RADS

Liver Imaging Reporting & Data Systems

LC-MS

Liquid chromatography–mass spectrometry

mRECIST

Modified Response Evaluation Criteria in Solid Tumors

N-P-gp

N-terminal of P-glycoprotein

OCR

Oxygen consumption rate

O-GlcNAc

O-GlcNAcylation

OTX

OTX008

PEP

Phosphoenolpyruvate

3/2PG

3/2-Phosphoglycerate

PGK

Phosphoglycerate kinase

TCA

Tricarboxylic acid

Wort

Wortmannin