Methods and compositions for cancer detection and therapy

Abstract

Aspects of the disclosure relate to the discovery that galectin-1 is increased in thermal-resistant cancers, including hepatocellular carcinoma. Certain aspects relate to methods of measuring galectin-1 levels to determine a patient's responsiveness to therapies, such as thermal ablation therapy. Certain aspects relate to inhibiting galectin-1 to improve responsiveness to a therapy, such as a thermal ablation therapy.

Description

METHODS AND COMPOSITIONS FOR CANCER DETECTION AND THERAPY

[0001] This application claims priority of U.S. Provisional Application No. 63 / 695,161 filed September 16, 2024, which is hereby incorporated by reference in its entirety.BACKGROUNDI. Field of the Invention

[0002] This disclosure relates to the fields of oncology, biochemistry, and medicine.II. Background

[0003] The metabolic heterogeneity of hepatocellular carcinoma (HCC) significantly contributes to therapeutic resistance, particularly in thermal ablation, a standard of care for early-stage and non-surgical patients. Approximately 80% of patients require adjuvant treatments following technically-successful ablations. While ablation can lead to cellular death in the central-treatment zone, its metabolic impact on peripheral cells remains unclear.BRIEF SUMMARY

[0004] In general, the current invention relates to the discovery that galectin-1 expression correlates with resistance to thermal ablation and tumor aggressiveness. This discovery can be utilized, in some aspects, to determine whether a patient will respond to a therapy and / or the aggressiveness of the cancer. Also, in some aspects, a galectin-1 inhibitor can be administered to a patient to increase responsiveness to a thermal ablation therapy and / or reduce tumor aggressiveness.

[0005] Disclosed herein are methods of treating hepatocellular carcinoma in a patient. The method can comprise one or more steps including any of: administering a galectin-1 inhibitor the patient, measuring a galectin-1 gene product, providing a thermal ablation therapy, administering a second therapy, and administering a pharmaceutical composition comprising a galectin-1 inhibitor. Also disclosed are methods of treating a thermal ablation-refractory hepatocellular carcinoma, methods of reducing tumor aggressiveness in a hepatocellular carcinoma patient, methods of sensitizing hepatocellular carcinoma to a thermal ablation therapy, and methods of killing hepatocellular carcinoma cells in a patient.299642387.1 - 1 -

[0006] In some aspects, patient has received thermal ablation therapy. In some aspects, the patient is indicated to receive thermal ablation therapy. In some aspects, the patient is determined to have an amount of a galectin- 1 gene product, in cancer cells from the patient, higher than a control. In some aspects, the control is an amount of a galectin- 1 gene product in cancer cells from a patient determined to be responsive to a thermal ablation therapy. In some aspects, the control is an amount of a galectin- 1 gene product in hepatocellular carcinoma cells from a patient determined to be responsive to a thermal ablation therapy. In some aspects, the control comprises an amount of galectin- 1 gene product in non-cancerous cells. In some aspects, the non-cancerous cells are liver cells. In some aspects, the amount of the galectin- 1 gene product is determined to be at least two-fold higher than the control. In some aspects, the amount of the galectin- 1 gene product is determined to be at least nine-fold higher than the control. In some aspects, the galectin-1 inhibitor is OTX008. In some aspects, the galectin-1 inhibitor comprises a nucleic acid capable of reducing a galectin-1 mRNA gene product. In some aspects, the galectin-1 inhibitor comprises a galectin-1 mRNA-targeting siRNA. In some aspects, the galectin-1 inhibitor comprises a galectin-1 binding protein. In some aspects, the galectin-1 binding protein comprises a galectin-1 antibody. In some aspects, the galectin-1 binding protein comprises a carbohydrate, peptide, peptidomimetic, or analog thereof. In some aspects, the galectin-1 inhibitor comprises a galectin-1 targeting gene therapy vector. It is also specifically contemplated that the galectin-1 inhibitor is not one of the inhibitors described above. In some aspects, the galectin-1 inhibitor is capable of reducing the amount of a galectin- 1 gene product in a cancer cell in the patient. In some aspects, the patient is a human patient.

[0007] Also disclosed are methods of measuring a gene profile in a hepatocellular carcinoma patient. The methods can comprise one or more steps including any of: measuring an amount of a galectin- 1 gene product in cancer cells from the patient, isolating nucleic acids and / or proteins from a biological sample from the patient, and obtaining a biological sample from a patient.

[0008] In some aspects, patient has received thermal ablation therapy. In some aspects, the patient is indicated to receive thermal ablation therapy. In some aspects, the method further comprises comparing the measured amount of the galectin-1 gene product to a control. In some aspects, the method further comprises providing an ablation therapy to the patient when the amount of the galectin-1 gene product is lower than the control. In some aspects, the control is an amount of a galectin-1 gene product in cancer cells from a patient determined to be responsive to a thermal ablation therapy. In some aspects, the control is an amount of a galectin-1 gene product in hepatocellular carcinoma cells from a patient determined to be299642387.1 - 2 -responsive to a thermal ablation therapy. In some aspects, the control comprises an amount of galectin-1 gene product in non-cancerous cells. In some aspects, the non-cancerous cells are liver cells. In some aspects, the non-cancerous cells are taken from the patient. In some aspects, the method further comprises administering a galectin-1 inhibitor to the patient. In some aspects, the patient is a human patient.

[0009] Also disclosed are methods of determining tumor aggressiveness in a patient, methods of determining responsiveness to a thermal ablation therapy in a patient, methods of prognosing a patient, methods of determining a treatment plan for a patient, and methods of diagnosing a patient with refractory cancer. The methods can comprise one or more steps including one or more of the steps disclosed herein.

[0010] In some aspects, the patient has, has been diagnosed with, has symptoms of, is suspected of having hepatocellular carcinoma.

[0011] Also disclosed are one or more of the following enumerated Aspects:Aspect 1. A method of treating hepatocellular carcinoma in a patient, the method comprising administering a galectin- 1 inhibitor the patient.Aspect 2. A method of reducing tumor aggressiveness in a hepatocellular carcinoma patient, the method comprising administering a galectin-1 inhibitor to the patient.Aspect 3. The method of Aspect 1 or 2, wherein patient has received thermal ablation therapy.Aspect 4. The method of any one of Aspects 1 to 3, wherein the patient is indicated to receive thermal ablation therapy.Aspect 5. The method of any one of Aspects 1 to 4, wherein the patient is determined to have an amount of a galectin-1 gene product, in cancer cells from the patient, higher than a control.Aspect 6. The method of Aspect 5, wherein the control is an amount of a galectin-1 gene product in cancer cells from a patient determined to be responsive to a thermal ablation therapy.Aspect 7. The method of Aspect 5, wherein the control is an amount of a galectin-1 gene product in hepatocellular carcinoma cells from a patient determined to be responsive to a thermal ablation therapy.Aspect 8. The method of Aspect 5, wherein the control comprises an amount of galectin-1 gene product in non-cancerous cells.299642387.1 - 3 -Aspect 9. The method of Aspect 8, wherein the non-cancerous cells are liver cells.Aspect 10. The method of any one of Aspects 5 to 9, wherein the amount of the galectin-1 gene product is determined to be at least two-fold higher than the control.Aspect 11. The method of any one of Aspects 5 to 9, wherein the amount of the galectin-1 gene product is determined to be at least nine-fold higher than the control.Aspect 12. The method of any one of Aspects 1 to 11, wherein the galectin-1 inhibitor comprises a nucleic acid capable of reducing a galectin-1 mRNA gene product.Aspect 13. The method of any one of Aspects 1 to 11, wherein the galectin-1 inhibitor comprises a galectin-1 mRNA-targeting siRNA.Aspect 14. The method of any one of Aspects 1 to 11, wherein the galectin-1 inhibitor comprises a galectin-1 binding protein.Aspect 15. The method of Aspect 14, wherein the galectin-1 binding protein comprises a galectin-1 antibody.Aspect 16. The method of Aspect 14, wherein the galectin-1 binding protein comprises a carbohydrate, peptide, peptidomimetic, or analog thereof.Aspect 17. The method of any one of Aspects 1 to 11, wherein the galectin-1 inhibitor comprises a galectin-1 targeting gene therapy vector.Aspect 18. The method of any one of Aspects 1 to 17, wherein the galectin-1 inhibitor is capable of reducing the amount of a galectin- 1 gene product in a cancer cell in the patient.Aspect 19. The method of any one of Aspects 1 to 18, wherein the patient is a human patient.Aspect 20. A method of measuring a gene profile in a hepatocellular carcinoma patient, the method comprising measuring an amount of a galectin- 1 gene product in cancer cells from the patient.Aspect 21. The method of Aspect 20, wherein patient has received thermal ablation therapy.Aspect 22. The method of Aspect 20 or 21, wherein the patient is indicated to receive thermal ablation therapy.Aspect 23. The method of any one of Aspects 20 to 22, further comprising comparing the measured amount of the galectin-1 gene product to a control.299642387.1 - 4 -Aspect 24. The method of any one of Aspects 20 to 23, further comprising providing an ablation therapy to the patient when the amount of the galectin-1 gene product is lower than the control.Aspect 25. The method of Aspect 24, wherein the control is an amount of a galectin-1 gene product in cancer cells from a patient determined to be responsive to a thermal ablation therapy.Aspect 26. The method of Aspect 24, wherein the control is an amount of a galectin-1 gene product in hepatocellular carcinoma cells from a patient determined to be responsive to a thermal ablation therapy.Aspect 27. The method of Aspect 24, wherein the control comprises an amount of galectin-1 gene product in non-cancerous cells.Aspect 28. The method of Aspect 27, wherein the non-cancerous cells are liver cells.Aspect 29. The method of Aspect 27 or 28, wherein the non-cancerous cells are taken from the patient.Aspect 30. The method of any one of Aspects 20 to 29, further comprising administering a galectin-1 inhibitor to the patient.Aspect 31. The method of any one of Aspects 20 to 30, wherein the patient is a human patient.Aspect 32. A method of determining tumor aggressiveness and / or responsiveness to a thermal ablation therapy in a hepatocellular carcinoma patient, the method comprising measuring an amount of a galectin- 1 gene product in cancer cells from the patient.Aspect 33. The method of Aspect 32, wherein patient has received thermal ablation therapy.Aspect 34. The method of Aspect 32 or 33, wherein the patient is indicated to receive thermal ablation therapy.Aspect 35. The method of any one of Aspects 32 to 34, further comprising comparing the measured amount of the galectin-1 gene product to a control.Aspect 36. The method of any one of Aspects 32 to 35, further comprising providing an ablation therapy to the patient when the amount of the galectin-1 gene product is lower than the control.299642387.1 - 5 -Aspect 37. The method of Aspect 36, wherein the control is an amount of a galectin-1 gene product in cancer cells from a patient determined to be responsive to a thermal ablation therapy.Aspect 38. The method of Aspect 36, wherein the control is an amount of a galectin-1 gene product in hepatocellular carcinoma cells from a patient determined to be responsive to a thermal ablation therapy.Aspect 39. The method of Aspect 36, wherein the control comprises an amount of galectin-1 gene product in non-cancerous cells.Aspect 40. The method of Aspect 39, wherein the non-cancerous cells are liver cells.Aspect 41. The method of Aspect 39 or 40, wherein the non-cancerous cells are taken from the patient.Aspect 42. The method of any one of Aspects 32 to 41, further comprising administering a galectin-1 inhibitor to the patient.Aspect 43. The method of any one of Aspects 32 to 42, wherein the patient is a human patient.

[0012] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0013] As used herein, “isolated” means altered or removed from the natural state through human intervention.

[0014] As used herein, the terms “therapeutic composition,” “pharmaceutical composition,” “therapeutic agent” and “pharmaceutical agent” may be used interchangeably and refer to a composition that is used therapeutically to affect a response in a patient.

[0015] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Any term used in singular form also comprises plural forms and vice versa.

[0016] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an aspect or aspect.299642387.1 - 6 -

[0017] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0018] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. The phrase “consisting of’ excludes any element, step, or ingredient not specified. The phrase “consisting essentially of’ limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments and aspects described in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”

[0019] It is contemplated that any aspect discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0020] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0021] Use of the one or more sequences or compositions may be employed based on any of the methods described herein. Other aspects and embodiments are discussed throughout this application. Any embodiment or aspect discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa.

[0022] It is specifically contemplated that any limitation discussed with respect to one embodiment or aspect of the invention may apply to any other embodiment or aspect of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also aspects that may be implemented in the context of aspects discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of Invention, Detailed Description of the Embodiments, Claims, and description of Figure Legends.299642387.1 - 7 -

[0023] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0025] FIG. 1A shows a Kaplan-Meier survival curve on thermal- ablation responders versus non-responders.

[0026] FIG. IB shows a heatmap representing abundance of galectin-1, glucose-6- phosphate, phosphor-glycerate kinase, and glyceraldehyde 3P dehydrogenase in FFPE biopsy specimens of responders and non-responders.

[0027] FIG. 1C is a graphical representation of galectin-1 abundance in biopsy specimens of responders and non-responders.

[0028] FIG. ID is a graphical representation of glucose-6P isomerase abundance in biopsy specimens of responders and non-responders.

[0029] FIG. IE is a graphical representation of phosphoglycerate kinase abundance in biopsy specimens of responders and non-responders.

[0030] FIG. IF is a graphical representation of GADPH abundance in biopsy specimens of responders and non-responders.

[0031] FIG. 2A is a graphical representation of percentages of SNU-449 and SNU423 cells relative to control post 47°C thermal exposure over time.

[0032] FIG. 2B is a graphical representation of SNU-423 and SNU-449 cell numbers post 37°C and 47°C thermal exposure over time.

[0033] FIG. 2C shows bright-field microscopy images of SNU-449 and SNU-423 cells post thermal exposure over time.

[0034] FIG. 2D is an image of a Western blot of galectin-1 in SNU-449, SNU-423, and HepG2 / C3a cells.299642387.1 - 8 -

[0035] FIG. 3A is a graphical representation of cell numbers of SNU-449 post hyperthermic exposure, with or without gal-1 inhibitor OTX treatments, over time.

[0036] FIG. 3B is a graphical representation of percentage of SNU-449 cells relative to control, post 47°C thermal exposure with gal-1 inhibitor, OTX, over time.

[0037] FIG. 3C is an image of a Western blot for gal-1 in SNU-449 cells transduced with lentiviral particles carrying control shRNA and gal-1 shRNA.

[0038] FIG. 3D is a graphical representation of relative gal-1 expression in cells transduced with lentiviral particles carrying control shRNA and gal-1 shRNA.

[0039] FIG. 3E is a graphical representation of numbers of transduced SNU-499 cells post hyperthermic exposure over time.

[0040] FIG. 3F is a graphical representation of percentage of transduced SNU-499 cells relative to control post 37°C and 47°C exposure over time.

[0041] FIG. 4A is a graphical representation of percentage of ATP relative to total ATP in SNU-499 wildtype cells in presence or absence of OTX.

[0042] FIG. 4B shows an image of a Western blot for gal-1 and P-glycoprotein bound to gal-1 in SNU-499 wildtype cells.

[0043] FIG. 4C is a graphical representation of relative gal-1 expression in SNU-499 wildtype cells that were exposed to 37°C or 47°C.

[0044] FIG. 4D is a graphical representation of relative P-gp bound gal-1 expression in SNU-499 wildtype cells that were exposed to 37°C or 47°C.

[0045] FIG. 4E is a graphical representation of percentage ATP relative to total ATP in SNU423 wildtype cells in presence or absence of OTX.

[0046] FIG. 4F shows an image of a Western blot for gal-1 P-gp bound gal-1 in SNU-423 wildtype cells.

[0047] FIG. 4G is a graphical representation of relative gal-1 expression in SNU-423 cells that were exposed to 37°C or 47°C.

[0048] FIG. 4H is a graphical representation of relative P-gp bound gal-1 expression in SNU-423 wildtype cells that were exposed to 37°C or 47°C.

[0049] FIG. 5 A is a schematic diagram depicting an interaction between gal-1, GM1 ganglioside, P-galactosidase, and P-glycoprotein.

[0050] FIG. 5B shows Western blot images of N-P-gp / gal- 1 bound GM 1 level with DMSO or OTX treatment.

[0051] FIG. 5C is a graphical representation of GM1 protein abundance levels in cells withDMSO or OTX treatment.299642387.1 - 9 -

[0052] FIG. 5D is a graphical representation of P-galactosidase activity in SNU-423 and SNU-499 cells with DMSO or OTX treatment.

[0053] FIG. 5E shows Western blot images of N-P-glycoprotein bound galactosidase levels in SNU-423 and SNU-499 cells with DMSO or OTX treatment.

[0054] FIG. 5F is a graphical representation of P-galactosidase and N-P-glycoprotein bound P-galactosidase levels in SNU-423 and SNU-499 cells with DMSO or OTX treatment.

[0055] FIG. 6A is a schematic diagram depicting a hydrolysis reaction of GM1 bound gal- 1.

[0056] FIG. 6B is a graphical representation of P-galactosidase activity of SNU-499 cells that were transduced with lentivirus particles carrying shGal-1 or shControl.

[0057] FIG. 6C shows Western blot images of N-P-glycoprotein / Gal-1 bound GM1 levels in cells that were transduced with lentivirus particles carrying shGal-1 or shControl.

[0058] FIG. 6D is a graphical representation of N-P-glycoprotein / Gal-1 bound GM1 levels in cells that were transduced with lentivirus particles carrying shGal-1 or shControl.

[0059] FIG. 6E is a graphical representation of galactose intensity in cells that were transduced with lentivirus particles carrying shGal-1 or shControl.

[0060] FIG. 6F shows Western blot images of N-P-glycoprotein bound P-galactosidase levels in cells that were transduced with lentivirus particles carrying shGal-1 or shControl.

[0061] FIG. 6G is a graphical representation of N-P-glycoprotein bound P-galactosidase abundance in cells that were transduced with lentivirus particles carrying shGal-1 or shControl.

[0062] FIG. 6H is a graphical representation of glycolytic ATP production in cells that were transduced with lentivirus particles carrying shGal-1 or shControl.

[0063] FIG. 7 is a schematic diagram depicting the interactions between P-glycoprotein, P- galactosidase, and gal-1.

[0064] FIG. 8A is a schematic diagram of Gal- 1 -mediated glycolytic pathway.

[0065] FIG. 8B shows Western blot images of N-P-gp / Gal bound GM1 levels in cells exposed to 37°C and 47°C, that were transduced with lentivirus particles carrying shControl, shCTR, or shGal- 1.

[0066] FIG. 8C is a graphical representation of normalized galactose intensity of cells exposed to 37 °C or 47 °C, that were transduced with lentivirus particles carrying shControl or shGal- 1.

[0067] FIG. 8D is a graphical representation of glycolytic activities of cells exposed to 37°C or 47°C, that were transduced with lentivirus particles carrying shControl or shGal-1.

[0068] FIG. 9 is a schematic diagram of13C-isotope label tracing.299642387.1 - 10 -

[0069] FIG. 10A is a schematic diagram of tumor implantation and treatments.

[0070] FIG. 10B is a graphical representation of tumor weights of tumors from animals that received DMSO, thermal-ablation treatment and DMSO, OTX, or thermal- ablation treatment and OTX.

[0071] FIG. 10C shows images of liver orthotopic SNU-499 tumors from animals that received DMSO, thermal-ablation treatment and DMSO, OTX, or thermal-ablation treatment and OTX.

[0072] FIG. 10D shows Western blot images for hexokinase II, PDH, LDHA, Gal-1, and P-Tubulin levels in SNU-499 tumors from animals that received DMSO, thermal- ablation treatment and DMSO, OTX, or thermal- ablation treatment and OTX.

[0073] FIG. 11 A depicts a hyperthermic exposure setup.

[0074] FIG. 1 IB is a graphical representation of SNU423 cell numbers after treatment with 0 pM, 25 pM, 50 pM, 75 pM, or 100 pM of OTX, over time.

[0075] FIG. 11C is a graphical representation of SNU-449 cell numbers after treatment with 0 pM, 25 pM, 50 pM, 75 pM, or 100 pM of OTX, over time.

[0076] FIG. 1 ID is a graphical representation of percentage of SNU-423 cells relative to control at 37°C or 47°C with DMSO or OTX, over time.

[0077] FIG. 12A is a graphical representation of normalized galactose intensity in SNU- 423 and SNU-449 cells.

[0078] FIG. 12B shows Western blot images of P-glycoprotein expression in SNU-423 and SNU-449 cells.

[0079] FIG. 12C shows orthotopic SNU-449 tumor volume of tumors from animals that received DMSO, thermal-ablation treatment and DMSO, OTX, or thermal-ablation treatment and OTX.

[0080] FIG. 12D is a graphical representation of SNU-449 cell numbers with DMSO or 50 pM Gal-1 inhibitor treatment at 0 and 24 hour timepoints.

[0081] FIG. 12E is a graphical representation of percentage glycolytic ATP relative to total ATP of SNU-449 with DMSO or 50 pM Gal-1 inhibitor treatment.

[0082] FIG. 12F shows Western blot images of GAPDH, PFK1, and P-Tubulin in SNU- 449 tumors from animals that received DMSO, thermal-ablation treatment and DMSO, OTX, or thermal- ablation treatment and OTX.299642387.1 - 11 -DETAILED DESCRIPTION

[0083] Thermal ablation is a minimally invasive procedure used to treat unresectable early - stage hepatocellular carcinoma. While the procedure is considered safe and as efficacious as surgery, there is a risk for rapid recurrence and progression in a subset of patients. This can result in additional therapies and may even move the patient off of the transplant list. Aspects herein relate to a key protein that is over expressed in this specific high-risk patient population. Aspects herein relate to the modulation of this protein, either through pharmacologic, genetic, or immunologic methods, which can decrease the aggressiveness of this tumor profile. Modulating this protein in an adjuvant manner can help improve the clinical outcomes in patients undergoing thermal ablation therapy. Another aspect for this protein is as a blood or tissue biomarker to stratify tumor aggressiveness, and help assign patients to specific locoregional or systemic therapies. For example, high expression of this marker can serve as a biomarker to assign a patient to ablation plus systemic therapy at an earlier time point.I. Administration of Therapeutic Compositions

[0084] The therapy provided herein may comprise the administration of one or a combination of therapeutic agents, such as one or a combination of galectin-1 inhibitors, including any galectin-1 inhibitors described herein, and other therapeutic compositions, including those useful for treating disorders disclosed herein, such as any cancer or disease associated with galectin-1, to a patient. In some aspects, the therapeutic agent is OTX008. In some aspects, the therapeutic agent is a single antisense oligonucleotide (ASO) or a cocktail of ASOs, including ASOs that bind to and decrease a galectin-1 gene product. In some aspects, the other therapeutic agents are useful for reducing symptoms of the disease and / or reducing side effects of the other therapeutic agents administered. The therapies may be administered in any suitable manner known in the art. In some aspects, a first therapeutic composition (such as a first galectin-1 inhibitor) and a second composition (such as another galectin-1 inhibitor or another therapeutic composition) may be administered sequentially (at different times) or concurrently (at the same time). In some aspects, the first and second therapeutic compositions are administered in a separate composition. In some aspects, the first and second therapeutic compositions are in the same composition.

[0085] In some aspects, the first therapeutic composition and the second therapeutic composition are administered substantially simultaneously. In some aspects, the first therapeutic composition and the second therapeutic composition are administered sequentially.299642387.1 - 12 -In some aspects, the first therapeutic composition, the second therapeutic composition, and a third therapeutic composition are administered sequentially. In some aspects, the first therapeutic composition is administered before administering the second therapeutic composition. In some aspects, the first therapeutic composition is administered after administering the second therapeutic composition.

[0086] In some aspects, thermal ablation is provided to the patient before, during, and / or after the administration of the therapeutic agent(s).

[0087] Aspects of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed.

[0088] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some aspects, the therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0089] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some aspects, a unit dose comprises a single administrable dose.

[0090] In some aspects, a single dose of the therapeutic composition is administered. In some aspects, multiple doses of the composition are administered. In some aspects, the therapeutic composition is administered at a dose of between 1 mg / kg and 5000 mg / kg. In some aspects, the therapeutic composition is administered at a dose of at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,299642387.1 - 13 -123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274,275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293,294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331,332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407,408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445,446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464,465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483,484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502,503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521,522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540,541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559,560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 mg / kg.

[0091] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain aspects, it is contemplated that doses in the range from 10 mg / kg to 200 mg / kg can affect the protective capability of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400,299642387.1 - 14 -500, 1000 pg / kg, mg / kg, g / day, or mg / day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and / or on multiple days, weeks, or months.

[0092] In certain aspects, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 pM to 150 pM. In another aspect, the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10 pM to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to 50 pM; or about 50 pM to 150 pM; or about 50 pM to 100 pM (or any range derivable therein). In other aspects, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 pM or any range derivable therein. In certain aspects, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0093] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0094] It will be understood by those skilled in the art and made aware that dosage units of pg / kg or mg / kg of body weight can be converted and expressed in comparable concentration units of pg / ml or mM (blood levels). It is also understood that uptake is species and organ / tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.299642387.1 - 15 -

[0095] In certain instances, it will be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, or 12 day, week, month, or year intervals, including all ranges there between.

[0096] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.

[0097] The active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0098] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0099] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars299642387.1 - 16 -or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum mono stearate and gelatin.

[0100] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0101] Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.

[0102] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactic ally effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.A. Therapuetic Compositions

[0103] In certain aspects, the compositions or agents, including those for use in the methods disclosed herein are suitably contained in a pharmaceutically acceptable carrier. The carrier can be non-toxic, biocompatible, and selected so as not to detrimentally affect the biological activity of the agent. The agents in some aspects of the disclosure may be formulated into preparations for local delivery (i.e. to a specific location of the body, such as the brain, nervous tissue, or other tissue) or systemic delivery, in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parenteral or surgical administration. Certain aspects of the299642387.1 - 17 -disclosure also contemplate local administration of the compositions by coating medical devices and the like.

[0104] Suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

[0105] The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.

[0106] In certain aspects, the actual dosage amount of a composition administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0107] Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0108] In certain aspects, the pharmaceutical compositions are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable or solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg or less, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.299642387.1 - 18 -

[0109] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antgifungal agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.

[0110] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[0111] In further aspects, the pharmaceutical compositions may include classic pharmaceutical preparations. Administration of pharmaceutical compositions according to certain aspects may be via any common route so long as the target tissue is available via that route. This may include oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. For treatment of conditions of the lungs, aerosol delivery can be used. Volume of the aerosol may be between about 0.01 ml and 0.5 ml, for example.

[0112] An effective amount of the pharmaceutical composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.

[0113] Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.299642387.1 - 19 -B. Proteins and Peptides

[0114] In certain aspects, herein the pharmaceutical composition comprises a protein. In certain aspects herein, the pharmaceutical composition comprises a In certain embodiments the size of a peptide or protein of the disclosure may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 amino acid residues or greater, and any range derivable therein. It is contemplated that polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.). As used herein, the term “domain” refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids with a structure or function recognizable by one skilled in the art.

[0115] It is contemplated that in compositions of the disclosure, including pharmaceutical compositions, there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and / or protein per ml. The concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein).C. Inhibitory Oligonucleotides

[0116] In some aspects, the disclosure relates to inhibitory oligonucleotides that inhibit the gene expression of galectin-1. Examples of an inhibitory oligonucleotides include but are not limited to siRNA (small interfering RNA), short hairpin RNA (shRNA), double-stranded RNA, an antisense oligonucleotide, a ribozyme, and an oligonucleotide encoding any thereof. An inhibitory oligonucleotide may inhibit the transcription of a gene or prevent the translation of a gene transcript in a cell. An inhibitory oligonucleotide acid may be from 16 to 1000 nucleotides long, and in certain embodiments from 18 to 100 nucleotides long. The oligonucleotide may have at least or may have at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 50, 60, 70, 80, or 90 (or any range derivable therein) nucleotides. The oligonucleotide may be DNA, RNA, or a cDNA that encodes an inhibitory RNA.

[0117] As used herein, “isolated” means altered or removed from the natural state through human intervention. For example, an siRNA naturally present in a living animal is not299642387.1 - 20 -“isolated,” but a synthetic siRNA, or an siRNA partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated siRNA can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the siRNA has been delivered.

[0118] Inhibitory oligonucleotides are well known in the art. For example, siRNA and double-stranded RNA have been described in U.S. Patents 6,506,559 and 6,573,099, as well as in U.S. Patent Publications 2003 / 0051263, 2003 / 0055020, 2004 / 0265839, 2002 / 0168707, 2003 / 0159161, and 2004 / 0064842, all of which are herein incorporated by reference in their entirety.

[0119] Particularly, an inhibitory oligonucleotide may be capable of decreasing the expression of galectin-1 by at least 10%, 20%, 30%, or 40%, more particularly by at least 50%, 60%, or 70%, and most particularly by at least 75%, 80%, 90%, 95%, 99%, or 100% more or any range or value in between the foregoing.

[0120] In further embodiments, there are synthetic oligonucleotides that are galectin-1 inhibitors. An inhibitor may be between 17 to 25 nucleotides in length and comprises a 5’ to 3’ sequence that is at least 90% complementary to the 5’ to 3’ sequence of a mature galectin-1 mRNA. In certain embodiments, an inhibitor molecule is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. Moreover, an inhibitor molecule has a sequence (from 5’ to 3’) that is or is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary, or any range derivable therein, to the 5’ to 3’ sequence of a mature galectin-1 mRNA, particularly a mature, naturally occurring mRNA. One of skill in the art could use a portion of the probe sequence that is complementary to the sequence of a mature mRNA as the sequence for an mRNA inhibitor. Moreover, that portion of the probe sequence can be altered so that it is still 90% complementary to the sequence of a mature mRNA.

[0121] In some embodiments, the inhibitory oligonucleotide is an analog and may include modifications, particularly modifications that increase nuclease resistance, improve binding affinity, and / or improve binding specificity. For example, when the sugar portion of a nucleoside or nucleotide is replaced by a carbocyclic moiety, it is no longer a sugar. Moreover, when other substitutions, such a substitution for the inter-sugar phosphodiester linkage are made, the resulting material is no longer a true species. All such compounds are considered to be analogs. Throughout this specification, reference to the sugar portion of a nucleic acid species shall be understood to refer to either a true sugar or to a species taking the structural place of the sugar of wild type nucleic acids. Moreover, reference to inter-sugar linkages shall299642387.1 - 21 -be taken to include moieties serving to join the sugar or sugar analog portions in the fashion of wild type nucleic acids.

[0122] The present disclosure concerns modified oligonucleotides, i.e., oligonucleotide analogs or oligonucleosides, and methods for effecting the modifications. These modified oligonucleotides and oligonucleotide analogs may exhibit increased chemical and / or enzymatic stability relative to their naturally occurring counterparts. Extracellular and intracellular nucleases generally do not recognize and therefore do not bind to the backbone-modified compounds. When present as the protonated acid form, the lack of a negatively charged backbone may facilitate cellular penetration.

[0123] The modified intemucleoside linkages are intended to replace naturally-occurring phosphodiester-5’ -methylene linkages with four atom linking groups to confer nuclease resistance and enhanced cellular uptake to the resulting compound.

[0124] Modifications may be achieved using solid supports which may be manually manipulated or used in conjunction with a DNA synthesizer using methodology commonly known to those skilled in DNA synthesizer art. Generally, the procedure involves functionalizing the sugar moieties of two nucleosides which will be adjacent to one another in the selected sequence. In a 5’ to 3’ sense, an “upstream” synthon such as structure H is modified at its terminal 3’ site, while a “downstream” synthon such as structure Hl is modified at its terminal 5’ site.

[0125] Oligonucleosides linked by hydrazines, hydroxylamines, and other linking groups can be protected by a dimethoxy trityl group at the 5 ’-hydroxyl and activated for coupling at the 3 ’-hydroxyl with cyanoethyldiisopropyl-phosphite moieties. These compounds can be inserted into any desired sequence by standard, solid phase, automated DNA synthesis techniques. One of the most popular processes is the phosphoramidite technique. Oligonucleotides containing a uniform backbone linkage can be synthesized by use of CPG- solid support and standard nucleic acid synthesizing machines such as Applied Biosystems Inc. 380B and 394 and Milligen / B io search 7500 and 8800s. The initial nucleotide (number 1 at the 3’-terminus) is attached to a solid support such as controlled pore glass. In sequence specific order, each new nucleotide is attached either by manual manipulation or by the automated synthesizer system.

[0126] Free amino groups can be alkylated with, for example, acetone and sodium cyanoboro hydride in acetic acid. The alkylation step can be used to introduce other, useful, functional molecules on the macromolecule. Such useful functional molecules include but are not limited to reporter molecules, RNA cleaving groups, groups for improving the299642387.1 - 22 -pharmacokinetic properties of an oligonucleotide, and groups for improving the pharmacodynamic properties of an oligonucleotide. Such molecules can be attached to or conjugated to the macromolecule via attachment to the nitrogen atom in the backbone linkage. Alternatively, such molecules can be attached to pendent groups extending from a hydroxyl group of the sugar moiety of one or more of the nucleotides. Examples of such other useful functional groups are provided by WO1993007883, which is herein incorporated by reference, and in other of the above-referenced patent applications.

[0127] Solid supports may include any of those known in the art for polynucleotide synthesis, including controlled pore glass (CPG), oxalyl controlled pore glass, TentaGel Support — an aminopolyethyleneglycol derivatized support or Poros — a copolymer of polystyrene / divinylbenzene. Attachment and cleavage of nucleotides and oligonucleotides can be effected via standard procedures. As used herein, the term solid support further includes any linkers (e.g., long chain alkyl amines and succinyl residues) used to bind a growing oligonucleoside to a stationary phase such as CPG. In some embodiments, the oligonucleotide may be further defined as having one or more locked nucleotides, ethylene bridged nucleotides, peptide nucleic acids, or a 5’(E)-vinyl-phosphonate (VP) modification. In some embodiments, the oligonucleotides has one or more phosphorothioated DNA or RNA bases.D. Other Agents

[0128] It is contemplated that other agents may be used in combination with certain aspects of the present aspects to improve the therapeutic efficacy of treatment. These additional agents include agents that act in combination and / or synergistically with the ASOs described herein. The additional agents may comprise agents that reduce symptoms of the disorders disclosed herein, or may comprise agents that reduce side effects associated with the therapeutic compositions disclosed herein.Examples

[0129] The examples provided herein and in the Appendix are included to demonstrate certain aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which299642387.1 - 23 -are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.Example 1

[0130] Background: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer death worldwide. Thermal ablation has emerged as the standard of care for non-surgical HCC patients because of its similar efficacy to resection with fewer complications. However, more than 50% of ablation patients will have recurrence within 5 years - necessitating additional treatments. Recurrence rates are hypothesized to be related to underlying HCC biology. The goal of this study was to analyze the proteomic profiles of pre-ablation HCC biopsy samples and correlate proteomic changes to post- ablation outcomes. Proteomic markers of ablation resistance were validated in an in vitro model.

[0131] Methods: Clinical HCC biopsy samples were obtained prior to microwave ablation (UCLA IRB#23-000131). Recurrence rates in ablation responders (n=12) versus nonresponders (n=12) were then tracked for two years. Differential proteomic profiles acquired by LC-MS mass spectrometer were compared using biopsy samples between the responders and non-responders. The identified protein markers were confirmed with internally validated ablation-responsive and ablation-resistant HCC cells (SNU-423 and HepG2 / C3a, respectively) via Western Blotting. Additionally, these proteomic changes were shown to directly modulate thermal resistance in these cell lines via a pharmacological approach.

[0132] Results: Proteomic analysis of HCC biopsy samples revealed that galectin-class proteins were upregulated two-fold in ablation non-responders versus responders (p < 0.001). Additionally, galectin-class proteins were found to be upregulated nine-fold in thermally- resistant HepG2 / C3a. Subsequently, in-vitro studies using selective galectin inhibitors to target galectin protein increased thermal sensitivity in thermally-resistant HepG2 / C3a.

[0133] Conclusions: This study revealed a key correlation between elevation of galectin- class proteins and thermal resistance in HCC biopsy samples. Moreover, galectin-class proteins were found to play a modulatory role in thermal resistance. These findings can be leveraged to develop novel galectin-based therapies to increase the efficacy of thermal ablation in HCC patients.Example 2 - Evaluation of biopsy specimens from HCC patients and. in vitro thermal-ablation model

[0134] The clinical observation in HCC patients motivated the inventors to investigate why some HCC patients responded to thermal ablation while others did not, even when their tumors may share histopathological similarities. The inventors first wondered about the correlation299642387.1 - 24 -between thermal ablation responsiveness and tumor progression survival probabilities; hence, the inventors performed the Kaplan-Meier analysis on thermal- ablation responders versus nonresponders. This analysis revealed that the responders had a significantly longer progression- free duration compared to that of the non-responders (90.33±7.87 (median not reached) versus 46.52±8.74 months (median: 52.7 months), p=0.028) (FIG. 1A). Additionally, the one and three-year progression-free survival rates were significantly higher in the responders versus non-responders: 97.06% versus 87.50% and 90.32% versus 59.82% (p=0.028), respectively (FIG. 1A). As previously demonstrated that response differences in therapeutic treatments could be due to the heterogeneity of tumor microenvironments1, the inventors decided to uncover the proteome of these patients using their formalin-fixed paraffin embedded (FFPE) biopsy specimens. Of note, these patients were matched for their clinical baseline and tumor characteristics.

[0135] The inventors retrospectively utilized the FFPE biopsy specimens of the responders (n=34 patients) and non-responders (n=24 patients). After protein extraction, the protein samples were submitted to mass spectrometry for proteomic profiling. The proteomic analysis shown that there was a significant elevation of galectin-1 (gal-1) in the non-responders versus responders (p=0.0024) (FIGS. IB and 1C). Furthermore, this analysis revealed that various critical proteins involved in the glycolytic pathway — a main ATP-producing source in cancer, also known as the Warburg effect2— were also remarkably upregulated in the non-responders versus responders: glucose-6-phosphate isomerase (p=0.00017), phosphoglycerate kinase (p=0.0036), and glyceraldehyde- 3 -phosphate dehydrogenase (p=0.0048) (FIGS. IB, and 1D- 1F).

[0136] To further investigate the role of gal-1 along with other identified glycolytic proteins, the inventors simulated the responder and non-responder model by creating an in- vitro thermal- ablation model. This model was involved exposing cells to normothermic (37°C) versus hyperthermic (47°C) temperatures in a water bath (FIG. 11A). The temperature and thermal duration were chosen as shown because these conditions have been shown to sufficiently stress the cells without being lethal to them3,4. The inventors then performed a pilot study where various HCC cell lines (SNU387, SNU423, SNU449, SNU475, HepG2, and HepG2 / C3a) were exposed to normothermic (37°C) and hyperthermic temperatures (47°C) (FIG. 11B). The percentage of cellular death under hyperthermic conditions was then determined relative to respective normothermic controls at 24, 48, and 72 hours. This analysis showed that SNU449 cells were significantly more resistant to hyperthermia than SNU423299642387.1 - 25 -throughout the indicated study periods (FIG. 2A). Consistently, the cell growth rates dropped more precipitously in SNU423 versus SNU449 (FIG. 2B). Moreover, this cell growth reduction pattern was observed and documented in bright-field microscopy images taken at 24, 48, and 72 hours (FIG. 2C). After SNU423 and SNU449 were identified as thermal- ablation responsive and non-responsive cells, respectively, the inventors wondered whether the pattern expression of gal- 1 observed in the proteomic analysis of FFPE biopsy specimen would remain upheld in vitro. Therefore, the inventors checked for the expression of gal-1 in SNU423 and SNU449 in which gal-1 was observed to be significantly more upregulated in ablation- nonresponsive SNU449 versus ablation-responsive SNU423 (FIG. 2D). Of note, HepG2 / C3a was a positive control. Overall, these findings suggested the direct correlation between gal-1 levels and thermal- ablation responsiveness both in HCC patients and HCC-derived cell lines. Example 3 - Modulating galectin-1 for thermal- ablation non-responsiveness in vitro

[0137] To decipher the role of gal-1 in thermal-ablation non-responsiveness, a selective gal-1 inhibitor (OTX)5was utilized. Given the more abundance of gal-1 in SNU449 (FIG. 2D), the inventors decided to first perform the investigation in this cell line as it would allow us to better understand the role of gal-1. After being seeded in a 12- well plate for 24 hours, cells were treated with 50pM gal-1 inhibitor OTX for 24 hours prior to being exposed to 37°C or 47°C. Of note, the inventors chose 50pM dose for OTX because this dose was found to sufficiently reduce cell growth in the pilot studies for both SNU423 and SNU449 (FIGS. 11C- 11D) and in prior work5.The growth of those SNU449 cells exposed to 47°C with gal-1 inhibition was precipitously diminished compared to that of those only exposed to 47°C (FIG. 3A). Consistently, the percentage of cellular death in SNU449, calculated by dividing the number of dead cells by that of the respective controls, demonstrated a significant reduction in thermal- ablation resistance when cells were exposed to hyperthermic temperatures (47°C) with gal-1 inhibition (FIG. 3B). Similarly, the thermal- ablation responsive pattern in the presence of gal-1 inhibitor OTX was also observed in SNU423 (FIG. HE). In addition to pharmacologically demonstrating the association between thermal-ablation responsiveness and gal-1 levels, the expression of gal-1 was also partially silenced in SNU449 using lentiviral particles carrying shRNA. SNU449 cells were successfully reduced gal-1 expression by about 60% (FIGS. 3C-3D). shGal- 1 / 449 and respective shControl / 449 were tested with hyperthermic exposure at 47°C. The cell growth curve indicated that shGal- 1 / 449 at 47°C was significantly more responsive to thermal ablation than shControl at the same temperature (FIG. 3E). The cell survival percentage analysis also confirmed the results found in the cell growth curve (FIG. 3F). Collectively, these results suggested that high levels of gal-1 caused thermal-ablation non-299642387.1 - 26 -responsiveness in HCC cells, and that gal-1 expression regulated thermal-ablation responsiveness.Example 4 - Analysis of glycolytic activities

[0138] Given the proteomic findings in FFPE biopsy specimens of thermal- ablation responders versus non-responders in which glycolytic proteins were found to be significantly upregulated in the non-responder cohort (FIGS. 1D-1F). The inventors wondered whether glycolytic activities in thermal-ablation responsive versus non-responsive cells would also depend on gal-1 levels. To test this question, extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were assessed to indirectly evaluate the glycolytic and mitochondrial oxidative phosphorylation activities, respectively, by using a Seahorse real-time cell metabolic analyzer. This analysis was performed in both thermal- ablation non-responsive SNU449 and responsive SNU423. In SNU449, the analysis revealed that glycolytic activities were attenuated by inhibiting gal-1 via selective inhibitor OTX at 37°C (FIG. 4A). However, the glycolytic activities were upregulated under hyperthermic temperature at 47°C, which was conversely reduced in the presence of a selective gal-1 inhibitor at the same temperature. Notably, the expression of unbound gal-1 was upregulated under hyperthermia (FIGS. 4B- 4C). Another protein observed at 92kDa was also upregulated when cells were exposed to hyperthermic temperature at 47°C (FIGS. 4B and 4D). P-glycoprotein (P-gp) is an ATP- binding cassette (ABC) transporter composed of two halves: the N-terminal half (N-half) and the C-terminal half (C-half)6,7. These halves are connected by a flexible and thermal- sensitive linker7. P-gp is found in various intracellular compartments and can be oriented towards the cytoplasm6,8. P-gp also possesses P(l-4)-N-acetyl-D-glucosamine residues which predominantly bind to many mammalian galectins, including gal-1 that can be located in the cytoplasm9 10. Based on these findings, it is reasonable to conclude that the 92kDa band represents a complex formed by the N-half of P-gp binding to gal-1, which is located intracellularly. Consistently, the inventors observed these phenomena in the ablation- responsive SNU423 cells: upregulation of glycolysis under hyperthermia at 47°C (FIG. 4E) along with the upregulated expression of gal-1 and gal-1 bound to N-half of P-gp (FIGS. 4F- 4H). Altogether, these findings suggested that gal-1 levels were associated with glycolytic activities under normothermia (37°C) and hyperthermia (47°C), and strongly implicated the connection between gal-1 expression with glycolytic activities under hyperthermia at 47°C.Example 5 - Inhibition ofgalectin-1 in vitro

[0139] In addition to binding to N-half of P-gp, gal-1 has high affinity for and can be colocated intracellularly with GM1 (mono sialo tetrahexosylganglio side) ganglioside, a member299642387.1 - 27 -of glyco sphingolipid family containing sialic acid residues11 12. Moreover, -galactosidase (|3- gal / B-gal) has been shown to have high affinity for P(l-4)-N-acetyl-D-glucosamine residues at the N-half of P-gp (N-P-gp)13,14, which is the same binding site as gal-19,10. Based on these findings, the inventors reasoned that galectin-1 bound to N-terminal-half-P-glycoprotein intracellularly, which would allow galectin-1 to transport its bound GM1 ganglioside to P- galactosidase, a GM1 catalytic enzyme bound to another unoccupied N-terminal-half (FIG. 5A). To prove this mechanism of action of gal-1, a selective gal-1 inhibitor OTX was utilized to exert the inhibitory forces on the activities of gal-1, which was followed by staining for the total abundance of GM1 in the cell lysates using cholera toxin subunit B (CTBx)15. Upon gal- 1 inhibition, the abundance of GM 1 bound to the complex between N-P-gp and gal- 1 at 103kDa bands was highly increased on immunoblotting in both thermal-ablation responsive SNU423 and non-responsive SNU449 (FIGS. 5B-5C). As more GM1 remained intact upon gal-1 inhibition, the inventors then wondered whether the activity of -gal would be affected accordingly, meaning that P-gal would be less active when GM1 breakdown was reduced. Indeed, the activities of P-gal were significantly diminished in the presence of more GM1 upon gal-1 inhibition in both HCC cell lines (FIG. 5D). The inventors also aimed to demonstrate the mechanism by which P-gal exerted its catalytic action on GM1 breakdown. Consistent with previous work13,14, the inventors showed that P-gal bound to N-P-pg on immunoblotting at 193kDa (FIG. 5E). Additionally, the expression of this complex was noticed to be independent of gal-1 inhibition (FIG. 5F). Unbound P-gal expression was also not affected when gal-1 was inhibited (FIG. 5F). The unchanged expressions of unbound and N-P-pg bound P-gal in the context of P-gal’ s decreased activities suggested that low activity of P-gal enzyme was not due to its concentration but rather mainly due to the reduced availability of GM1 substrate upon blocking gal-l’s activity. Together, these results indicate the important role of gal-1 in facilitating the hydrolysis of GM1 via P-gal catalysis.Example 6 - Modulating [Egalactosidase activity in vitroModulating the activity of P-galactosidase directly affected abundance of GM1 bound to gal-1 and resulted in decreased glycolytic activity in HCC cells

[0140] To test whether reducing the activity of P-gal would directly affect the hydrolysis of GM1 bound to gal-1 (FIG. 6A), the inventors performed this investigation in gal-1 knockdown SNU449 (shGal- 1 / 449) cells compared to respective control (shControl / 449) cells. Without being limited by any theory, the inventors’ rationale was that because P-gal’ s activities had been demonstrated to be regulated by the PI3K signaling pathway whose phosphorylating299642387.1 - 28 -activation was directly controlled by the levels of gal-116 17; hence, if the inventors reduced gal- 1 expression via silencing its mRNA expression using lentiviral transfection approach, the inventors anticipated observing P-gal’s activities to reduce accordingly. The inventors found that P-gal was less active in shGal- 1 / 449 compared to that of shControl / 449 (FIG. 6B). Levels of GM1 bound to the N-P-gp / gal-1 complex were also noticed to change accordingly: more GM1 bound to gal-1 when |3-gal was less active in shGal-1 / 449 and less GM1 bound to gal-1 when P-gal was more active in shControl / 449 cells (FIGS. 6C-6D). As GM1 breakdown was less due to reduced activity of |3-gal in shGal- 1 / 449 cells, the galactose level was expected to go down in these cells. Furthermore, the level of galactose measured by fluorescence intensity was exhibited significantly less in the gal-1 KD SNU449 cells than that of their respective control cells (FIG. 6E). Of note, galactose level was also observed to reduce in gal-1- underexpressing SNU423 compared to that of gal-1 overexpressing SNU449 cells (FIG. 12A). Furthermore, the inventors confirmed the level of |3-gal bound to N-P-gp was higher when the enzyme was less active in catalyzing GM1 breakdown (FIGS. 6F-6G). This finding of increased |3-gal bound to N-P-gp was consistent with prior work because less gal-1 in KD cells would leave more unoccupied sites of N-P-gp for |3-gal to bind as they both shared high affinity for N-glycan terminal on P-gp9 10. The inventors then asked how the change in galactose concentration resulting from GM1 breakdown would affect cell energy production, particularly in glycolysis. The inventors utilized a Seahorse real-time cell metabolic analyzer to quantify the glycolytic activities in gal-1 KD SNU449 cells and respective control cells in which the inventors found that the glycolytic activities in gal-1 KD SNU449 cells were reduced as compared to that of the respective control cells (FIG. 6H). Consistently, the inventors noticed this glycolytic reduction pattern in gal- 1 -underexpressing SNU423 compared to that of gal-1- overexpressing SNU449 (FIGS. 4A and 4E). Therefore, these findings indicated that [3-gal modulated glycolytic levels via the hydrolysis of GM1 bound to gal-1, and suggested that modulating the activity of |3-galactosidase directly affected abundance of GM1 bound to gal-1 and resulted in decreased glycolytic activity in HCC cells.Example 7 - Downregulating P- glycoprotein expression

[0141] The inventors hypothesized that pharmacologically and genetically downregulating P-glycoprotein expression may attenuate the catalysis of Gal-1 bound GM1 and resultant glycolytic activity in HCC cells.299642387.1 - 29 -Example 8 - Metabolomics studies of cells under hyperthermia

[0142] The inventors hypothesized that metabolomics studies may demonstrate upregulation of glycolytic enzymes and metabolites under hyperthermia.Example 9 - Testing thermal ablation therapy in combination with a galectin-1 inhibitor

[0143] Given the discovery of this key metabolic adaptation mechanism in HCC cells subjected to ablation-induced hyperthermia, the inventors aimed to determine if targeting this pathway could improve the effectiveness of thermal ablation in reducing tumor size. The inventors selected thermal-ablation-non-responsive SNU449 cells for this in vivo orthotopic model. Success in controlling tumor progression with this more resistant cell line would further emphasize the critical role of this pathway in enabling resistant cells to develop metabolic plasticity for survival. After SNU449-cell derived tumors were orthotopically implanted into the murine livers, they were administered with selective gal-1 inhibitor OTX and then received thermal- ablation treatment as shown (FIG. 10A). Due to ethical guidelines, the tumors were harvested one week after thermal ablation, rather than being allowed to progress for a longer duration. Despite the short study duration, tumor weights were significantly lower in the group that received both thermal ablation and the treatment selective gal-1 inhibitor OTX (FIGS. 10B-10C). There was no reduction in tumor size in the group that received thermal ablation alone compared to the control group, as SNU449 cells have been shown to be highly resistant to thermal ablation (FIGS. 2A-2C). No reduction in tumor size was observed in the selective gal-1 inhibitor OTX alone group compared to the control, potentially due to the early termination of the experiment for ethical reasons. In addition to tumor weight, tumor volume measurements also revealed a significant reduction in the combined treatment group, despite no difference being noted in thermal ablation or OTX alone compared to the control group (FIG. 12C). The inventors then examined how these treatments altered the metabolic profiles of the tumors. As predicted by the in vitro findings, the thermal ablation alone group exhibited increased glycolytic activity compared to the control, as evidenced by a significant upregulation of hexokinase 2 and lactate dehydrogenase (FIG. 10D). The higher levels of pyruvate dehydrogenase, a key enzyme involved in oxidative phosphorylation, in the control group suggested lower glycolytic activity compared to the thermal ablation, OTX alone, or the combined group. However, glycolysis was also more upregulated in the gal-1 inhibitor OTX group compared to the control. Given that these mice received an inhibitor intended to decrease glycolytic activity, this finding may appear counterintuitive. However, the inventors speculated that inadequate inhibitor doses could have prompted tumor cells to upregulate glycolysis as a survival response. This phenomenon has been previously observed in vitro, where SNU449299642387.1 - 30 -cells treated with OTX exhibited increased glycolysis without dying (FIGS. 12D-12E). Notably, the thermal ablation and OTX alone groups exhibited an upregulation of gal-1 as compared to the control or the combined groups (FIG. 10D). This observation further supported the in vitro findings that cells would rely more on the complimentary galactose source to meet increased energy demand under stress. The inventors also examined other glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphofructokinase 1,2. While all groups showed increased glycolytic activity, no differential expression patterns were observed (FIG. 12F). These findings suggested that the efficacy of thermal ablation in tumor reduction was enhanced when combined with selective galectin- 1 inhibitor OTX.Example 10 - Methods and MaterialsPatient cohorts

[0144] This retrospective study was performed under written informed consents and approval from UCLA David Geffen School of Medicine Institutional Board Review (IRB#23- 000131). A total of n=58 previously banked needle biopsy samples were obtained from patients who had been diagnosed with moderate-differentiated (n=54) or poorly-differentiated (n=4) HCC. The responsiveness to thermal ablation from these patients was monitored for up to 9 years (108 months) with a median follow-up period of 38.12 months [interquartile range (IQR): 25.07 to 58.87 months]. Responders (n=34) were defined as those with tumors that were either decreasing in size or not increasing more than 20% of the original tumor size, for at least one year after the procedure. Conversely, non-responders (n=24) were those whose tumor progression exceeded 20% in size within one year following thermal ablation. Both study cohorts were matched for demographic, clinical baseline and tumor characteristics.Biopsy collection and FFPE preparation

[0145] Needle biopsy specimen from the liver were obtained directly from a Bard Mission 18-gauge spring loaded biopsy gun (BD, Franklin Lakes, NJ)18. These 2-cm cores were preserved using a standard protocol19. Specimens were immediately fixed with 10% formalin for 4- 24 hours to form cross-links and prevent degradation. After fixation, tissues were dehydrated by immersing in 100% ethanol (T038181000, ThermoFisher). Tissues were then cleared of the dehydrating 100% ethanol by incubating in 97% xylene (L13317.AP, ThermoFisher) before embedding. Paraffin was heated to 60oC and allowed to harden overnight. This processing protocol to produce FFPE blocks is the gold standard method to preserve human tissues for diagnosis or further disease characterization. Tissue blocks were sectioned in 5pm and mounted on a standard glass slide and covered with a slip cover.299642387.1 - 31 -Proteomic analysis

[0146] FFPE Sample Extraction & Preparation

[0147] 290 FFPE slices (n=5 per patient) with dimensions of 25x20x0.01 (mm) were scraped into centrifuge tubes and then de-paraffinized and de-crosslinked by heating. The samples were digested with trypsin. The digest was then run through a polar organic solvent mixture and centrifuged to remove undigested matrix. The supernatants were concentrated by SpeedVac drying and re-dissolving or stored at 4°C until analysis. A Thermo Vanquish UHPLC coupled to a Thermo Scientific Q Exactive Plus mass spectrometer via an electrospray ionization source was used for data acquisition. The sample extract was separated via the mixed mode liquid chromatography with mobile phase gradients composed of water, acetonitrile, and isopropanol, with formic acid & ammonium acetate mobile phase additives. Quality controls were incorporated into each batch to assess and correct for technical effects including QC pooled plasma, and QC water blank samples.

[0148] Identification: The collected peak fragmentation from the data dependent MS2 scans were matched to libraries of experimental and theoretical spectra. Moreover, identifications determined using Thermo Compound Discoverer mzCloud and the internal Dalton software with dot product > 80% and more than 3 matching fragmentation ions.

[0149] Relative quantification: Acquired raw MSI data were first aligned using persistent background ions as lock masses. MSI spectral correlations were then used to align MSI data retention time to a template run across the entire run period. MSI data were then calibrated for m / z (mass-to-charge ratio) based on all identified ions. Ion intensities were extracted by summing all ion signals within 5-ppm m / z and 1 -minute tolerance around the identified ions. Background ions were eliminated based on their presence in quality control water samples. Ions were aggregated into molecule level analytes by averaging. Outliers were omitted based on the correlation distance between each sample profile (r < 0.85). Intensity was normalized to correct for variable sample preparation and injection volumes by sample mass. Run order and batch effects were corrected by statistical linear modeling.Hyperthermic Resistance

[0150] The HCC cell lines SNU-423 and SNU-449 were procured from the American Type Culture Collection (ATCC). Cells were seeded at l.OxlO5cells in 12-well plates and cultured in RPMI containing 10% FBS, 4mM L-glutamine, 50Upenicillin / mL, and 50pg streptomycin / mL at 37°C with 5% CO2, for 24 hours. The 12-well plates were then be placed in a water bath at 37°C or 47°C for 10 minutes and allowed for rest at room temperature for 20 minutes. Cell death was monitored by counting using hemocytometer and 0.4% trypan blue299642387.1 - 32 -(cat# 15250061, ThermoFisher) at 24, 48, and 72 intervals. Of note, 47°C was used for the experiments as it had been demonstrated to be sublethal to HCC cells and allowed the cells to be able to recover from hyperthermia3,4. Percentage cell death was calculated by dividing the cell numbers from the experimental groups by the respective control groups. The cell growth bright-field images were captured using Keyence BZ-X series All-in-One fluorescence microscope at 24, 48, and 72 intervals prior to cell counting. Additionally, these experiments were performed using SNU-449 along with galectin-1 inhibition by a selective inhibitor OTX008 (HY-19756, Medchemexpress) and in shRNA transfected cells with galectin-1 knockdown. shRNA transfection in SNU-449 cells

[0151] SNU-449 cells were seeded at 3xl05cells in a 12-well plate to get to around 80% confluence the following day. Cells were cultured in RPMI containing 10% FBS, 4mM L- glutamine, 50Upenicillin / mL, and 50pg streptomycin / mL at 37°C. The following day, the cells were transduced with lentiviral shRNA particles carrying three to five expression constructs each encoding target- specific 19-25 nt (plus hairpin) shRNA designed to knockdown the gene expression of galectin-1 (sc-35441-V, scbt) or respective shControl lentiviral particles (sc- 108080, scbt). Briefly, ImL of RPMI mixed with Polybrene (sc- 134220, scbt) to get 5 pg / ml, was added each well of the 12-well plate after removing the old media. After that, IxlO6lentiviral particles were slowly added into the respective wells for shRNA or shControl groups. After 24 hours of transfection, the media was removed and replaced with complete RPMI containing 10% FBS, 50Upenicillin / mL, and 50pg streptomycin / mL and continued to incubate for another 24 hours. The following day (48 hours post transfection), the cells were split with a 1 / 3 ratio and continued with incubation for 24 hours. After that, the predetermined dose (15pg / mL) of puromycin (sc-108071, scbt) was added into each well to select for successfully- transduced cells. These cells were then checked for expression of Galectin-1 using western blot to ensure the success of transfection before utilizing the cells for experiments. The transfected cells were continued to be selected every 3 days with 15pg / mL puromycin.Galectin-1 overexpression using DNA plasmid

[0152] SNU-423 cells were seeded at 3xl05cells in a 12-well plate to get to around 80% confluence the following day. Cells were cultured in RPMI containing 10% FBS, 4mM L- glutamine, 50Upenicillin / mL, and 50pg streptomycin / mL at 37°C. Lipid-DNA complexes were prepared as follows. Briefly, tube 1 contained 50pL Opti-MEM medium (31985062, Thermo) and Lipofectamine™ 3000 (L3000015, Thermo), while tube 2 contained 50pL Opti-MEM medium and 6pL P3000 mixed with 3pL of Ipg / pL DNA plasmid pCMV6 vector encoded for299642387.1 - 33 -LGALS1 gene (SCI 18705, scbt) or equal amount of empty pCMV6 vector (PS 100001, scbt). Tube 2 was added into tube 1 and incubated for 10 minutes. This lOOpL mixture was then added into the respective wells, and the plate was gently swirled to ensure the complex was evenly distributed throughout each well. After 48 hours of transfection, the cells were split up with a 1 / 3 ratio and cultured in complete RPMI. The following day, cells were selected with a predetermined dose of 20mg / mL Neomycin (J6701 l.AD, Thermo). The cells were selected for 7 days and then harvested for western blot to ensure the success of galectin-1 overexpression. After that, the cells were expanded for experiments.Glycolytic activity / Oxygen consumption assessments

[0153] Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured using Seahorse XFe96 extracellular flux analyzer (Agilent Technologies) at UCLA Metabolism Core. Briefly, SNU-423 / SNU-449 / shGal-l / shControl SNU449 cells were seeded at the density of 20,000 cells per well in lOOpL of complete RPMI in the seahorse 96 well plate (102416-100, Agilent). After 24 hours of culturing, SNU-423 and SNU-449 cell were treated with a selective galectin-1 inhibitor OTX008 at a final concentration of lOOpM in each well. The cells were continued with incubation for another 24 hours. After that, these cells were exposed to 37°C or 47°C for 10 minutes and rested for 20 minutes at room temperature. After 3 hours of resting, cells were prepared for OCR and ECAR assessments. On the day of assay, the analyzer was prepared in accordance with the manufacturer’s recommended protocol. As the cell preparation, the cells were washed once with XF Real Time ATP Rate Assay Medium. The plate was then placed in a 37°C incubator without CO2 for one hour before running. The cartridge ports were loaded, and the assay was set up for running the assay. After the assay, the cells were stained using Ready Stains ( A49905, Fisher) for normalization.GM1 staining

[0154] After cells were subjected to appropriate experimental conditions, they were treated with cholera toxin subunit B (CTBx) (104, list labs) at the final concentration of 2pg / mL to stain for GM 1 as previously20. The cells were incubated with CTBx for 3 hour before harvesting for western blot. The harvested cells were then lysed and performed western blot with a standard protocol. The polyclonal CTBx antibody used in this staining experiment was Fisher Scientific (#22-704-0100UL) and was used at the dilution ratio of 1 / 500.Galactose concentration assessment

[0155] SNU423 / 449 / shControl-449 / shGal 1 -449 were seeded at 1 x 106cells in 10-cm dishes and cultured in RPMI containing 10% FBS, 4mM L-glutamine, 50Upenicillin / mL, and 50pg streptomycin / mL at 37°C. After 24 hours of culturing, SNU423 / 449 were treated with DMSO299642387.1 - 34 -vs OTX008 at lOOpM or DMSO vs GW5074 (HY- 10542, Medchemexpress) at 50pM. shControl-449 / shGal 1-449 were treated with DMSO vs GW5074 (HY-10542, Medchemexpress) at 50pM. After 24 hours of drug treatments, cells were harvested and counted to get lxl06per condition. Cells were then added lOOpL the lysis buffer from the galactose assay kit (ab83382, Abeam) and rapidly homogenized using sonicator. The mixture was rested on cold ice for 10 minutes before centrifuging at 13,000 rpm for 10 minutes to remove the insoluble materials. 50pL of each supernatant was added into each well of the 96- well black plate, which was followed by added 50pL of reaction mix containing the assay buffer, galactose probe, galactose enzyme mix, and developer solution / Horseradish peroxidase. The mixture was mixed well and incubated for 40 minutes at 22°C. The galactose standards were also prepared in accordance with manufacturer’s recommendations. The fluorescence intensity was then measured using SpectraMax M5 microplate reader at Excitation / Emission of 535 / 590nm.P-galactosidase activity assessment

[0156] SNU423 / 449 / shControl-449 / shGal 1 -449 were seeded at 1 x 106cells in 10-cm dishes and cultured in RPMI containing 10% FBS, 4mM L-glutamine, 50Upenicillin / mL, and 50pg streptomycin / mL at 37°C. After 24 hours of culturing, SNU423 and SNU449 were treated with DMSO or OTX008 at lOOpM. The following day, SNU423 and SNU449 treated with DMSO as vehicle controls or OTX008 were measure the activity of |3-galactosidase, whereas shControl-449 and shGal 1-449 were measured the activity of |3-galactosidasc after 24 hours of plating as follows. Briefly, the assay was purchased from Abeam (ab287846). Cells were first harvested and counted to get IxlO6cells for each group. Cells were then lysed using lOOpL of the P-gal assay buffer and rapidly homogenized by a sonicator. The homogenized mixture was rested on cold ice for 10 minutes. The supernatants were collected by centrifuging the mixture at 10,000g at 4°C for 5 minutes. 50pL of each supernatant was added into each desired well on a 96-well black plate. Positive controls were also made to ensure the quality of the readout data in accordance with manufacturer’s protocol. Each well was then added 50pL of a mixture containing [3-gal substrates. The fluorescence intensity data was recorded using SpectraMax M5 microplate reader at Excitation / Emission of 480 / 530nm in kinetic mode for 30 minutes at 37°C.Western Blotting and Brand Intensity Quantification

[0157] Cells were seeded at 4xl06in 10 cm dish and followed with appropriate experimental treatments: lOOpM OTX vs DMSO or 50pM GW5074 vs DMSO or non-299642387.1 - 35 -treatments. After 24 hours post treatments, cells were harvested and lysed using the lysis buffer containing 20mM Tris-HCl (15567027, Fisher) with lOpL / mL protease inhibitor cocktail (78440, Fisher). After vigorously sonicating, cells were rested in the lysis buffer for 10 minutes. The cells were then centrifuged at 17,000g for 5 minutes to collect the supernatant. The protein concentrations of the supernatants were then assessed using Pierce BCA Protein Assay kits (23227, Fisher) to ensure equal amounts of proteins being loaded into each well of the running gel. The supernatants were then added with equal volume of Bolts LDS Sample Buffer (B0008, Fisher) and heated up to 70°C, except for checking expression of p-glycoprotein as heating would disassociate this multiunit protein complex. After heating, appropriate amount of each supernatant was pipetted into a 2mL Eppendorf tube followed by lOpL of Bolt LDS sample buffer, 4pL of Bolt reducing agent (B0004, Fisher), deionized water such that the total volume was 40pL. Of note, the amount of each supernatant was calculated such that each well was loaded with 30pg protein. The gel was then run using MOPs (B0001, Fisher) if molecular weights (MWs) of desired proteins were 15kDa to 260kDa or MES (NP0002, Fisher) running buffer if MWs were from 3.5kDa to 160kDa. After running, the proteins from the gel were transferred to a membrane. The membrane was then blocked with Pierce Fast Blocking Buffer (37575, Fisher) for 30 minutes and incubated with primary antibodies overnight. The primary antibodies used in this study along with associated dilatation ratio were as follows: galectin-1 1 / 1000 (Abeam), |3-galactosidase 1 / 5000 (A-11132, Thermo), cholera toxin subunit B 1 / 500 (BS-12862R, Thermo), |3-tubulin 1 / 1000 (15568, Abeam). After primary antibody incubation, the membrane was washed with TBST for 3 times with 5 minutes each and was incubated with secondary antibody goat anti-rabbit IgG conjugated with Horseradish peroxidase at a 1 / 5000 dilution ratio for 1 hour. The membrane was then washed again with TBST for 3 times with 5 minutes each. Signals were developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (34580, Fisher) for 5 minutes. After that, the membrane was imaged using 1500 iBright imaging system. The brand intensity was also then quantified using Imagej software.Metabolomics labeling with6Ci3-isotope-labeled glucoseSNU-423 and SNU-449-cell-derived orthotopic implantation

[0158] WT SNU423 and SNU449 cells were mixed with complete RPMI containing 25% Matrigel (356234, Life Sciences) such that lOOpL volume contained IxlO6cells to inject subcutaneously into mouse flanks. After 2 weeks, tumors were excised from the flanks and orthotopically implanted into the liver following a standard protocol21. Briefly, 4- week-old nude mice were anesthetized using isoflurane, and the liver was exposed. A lOOmg tumor was299642387.1 - 36 -attached to the left-lateral lobe of the liver using 5.0 VICRYL suture (NC2872069, Fisher) and wrapped with a lx0.5-cm piece of SURGICEL (1951, Ethicon) to further protect the attachment and minimize bleeding. Incision was closed using wound clips (10-001-024, Fisher), and mice will be set up for recovery with a post-surgery care protocol21. After 2 weeks of implantation, the mice were randomly divided into 4 groups: control, ablation, OTX, ablation + OTX. Mice were then administered DMSO or OTX intraperitoneally at 5mg / kg5twice per week for 2 weeks. After that, ablation was performed in the respective groups by inserting a 14-gauge antenna to the center of tumors, which was connected to ECO Microwave Ablation (MWA) (ECO Inc., China) instrument to heat the tumors at 5W for 3 minutes22. After 30 minutes following the procedure, mice were administered with DMSO or OTX intraperitoneally at 5mg / kg and continued twice per week for another week. One week following ablation, tumors were harvested for weights, tumor volumes, and metabolomics.* * *

[0159] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred aspects, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.299642387.1 - 37 -REFERENCESThe references provided herein, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.1. Safri F, Nguyen R, Zerehpooshnesfchi S, George J, Qiao L. Heterogeneity of hepatocellular carcinoma: from mechanisms to clinical implications. Cancer Gene Ther. 2024 Mar 18;2. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009 May 22;324(5930): 1029-33.3. Obara K, Matsumoto N, Okamoto M, Kobayashi M, Ikeda H, Takahashi H, Katakura Y, Matsunaga K, Ishii T, Okuse C, Suzuki M, Itoh F. Insufficient radiofrequency ablation therapy may induce further malignant transformation of hepatocellular carcinoma. Hepatol Int. 2008 Mar;2(l): 116-23.4. Guo Y, Ren Y, Dong X, Kan X, Zheng C. An Overview of Hepatocellular Carcinoma After Insufficient Radiofrequency Ablation. JHC. 2022 Apr;Volume 9:343-55.5. Leung Z, Ko FCF, Tey SK, Kwong EML, Mao X, Liu BHM, Ma APY, Fung YME, Che CM, Wong DKH, Lai CL, Ng IOL, Yam JWP. Galectin-1 promotes hepatocellular carcinoma and the combined therapeutic effect of OTX008 galectin- 1 inhibitor and sorafenib in tumor cells. J Exp Clin Cancer Res. 2019 Oct 22;38(1):423.6. Fu D. Where is it and How Does it Get There - Intracellular Localization and Traffic of P- glycoprotein. Front Oncol [Internet]. 2013 [cited 2024 Aug 26] ;3. Available from: http: / / joumal.frontiersin.org / article / 10.3389 / fonc.2013.00321 / abstract7. Ward AB, Szewczyk P, Grimard V, Lee CW, Martinez L, Doshi R, Caya A, Villaluz M, Pardon E, Cregger C, Swartz DJ, Faison PG, Urbatsch IL, Govaerts C, Steyaert J, Chang G. Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain. Proc Natl Acad Sci USA. 2013 Aug 13; 110(33): 13386— 91.8. Han ES, Zhang JT. Mechanism Involved in Generating the Carboxyl-Terminal Half Topology of P-Glycoprotein. Biochemistry. 1998 Aug 1 ;37(34): 11996-2004.299642387.1 - 38 -9. Greer DA, Ivey S. Distinct N-glycan glycosylation of P-glycoprotein isolated from the human uterine sarcoma cell line MES-SA / Dx5. Biochimica et Biophy sica Acta (BBA) -General Subjects. 2007 Sep; 1770(9): 1275-82.10. Liu FT, Stowell SR. The role of galectins in immunity and infection. Nat Rev Immunol. 2023 Aug;23(8):479-94.11. Fajka-Boja R, Blaskd A, Kovacs-Sdlyom F, Szebeni GJ, Toth GK, Monostori E. Colocalization of galectin- 1 with GM 1 ganglioside in the course of its clathrin- and raft-dependent endocytosis. Cell Mol Life Sci. 2008 Aug;65(16):2586-93.12. Sipione S, Monyror J, Galleguillos D, Steinberg N, Kadam V. Gangliosides in the Brain: Physiology, Pathophysiology and Therapeutic Applications. Front Neurosci. 2020; 14:572965.13. Shimabuku AM, Nishimoto T, Ueda K, Komano T. P-glycoprotein. ATP hydrolysis by the N-terminal nucleotide-binding domain. J Biol Chem. 1992 Mar 5;267(7):4308-l l.14. Thoma J, Stenitzer D, Grabherr R, Staudacher E. Identification, Characterization, and Expression of a P-Galactosidase from Arion Species (Mollusca). Biomolecules. 2022 Oct 27;12(11):1578.15. Aman AT, Fraser S, Merritt EA, Rodigherio C, Kenny M, Ahn M, Hol WGJ, Williams NA, Lencer WI, Hirst TR. A mutant cholera toxin B subunit that binds GM1- ganglioside but lacks immunomodulatory or toxic activity. Proc Natl Acad Sci USA. 2001 Jul 17;98(15):8536— 41.16. Su YL, Luo HL, Huang CC, Liu TT, Huang EY, Sung MT, Lin JJ, Chiang PH, Chen YT, Kang CH, Cheng YT. Galectin- 1 Overexpression Activates the FAK / PI3K / AKT / mTOR Pathway and Is Correlated with Upper Urinary Urothelial Carcinoma Progression and Survival. Cells. 2020 Mar 26;9(4): 806.17. Di Blasio L, Puliafito A, Gagliardi PA, Comunanza V, Somale D, Chiaverina G, Bussolino F, Primo L. PI3K / mTOR inhibition promotes the regression of experimental vascular malformations driven by PIK3CA-activating mutations. Cell Death Dis. 2018 Jan 19;9(2):45.18. Tse JR, Terashima K, Shen L, McWilliams JP, Lu DSK, Raman SS. Safety of percutaneous, image-guided biopsy of hepatocellular carcinoma with and without concurrent ablation. Abdom Radiol (NY). 2022 Aug;47(8):2640-6.299642387.1 - 39 -19. O’Neil M, Damjanov I, Taylor RM. Handling and Processing of Liver Biopsies. In: Liver Pathology for Clinicians [Internet]. Cham: Springer International Publishing; 2015 [cited 2024 Jul 20]. p. 17-22. Available from: https: / / link.springer.eom / 10.1007 / 978-3-319-20080- 4_320. Watkins EB, Dennison AJC, Majewski J. Binding of Cholera Toxin B-Subunit to a Ganglioside GM 1 -Functionalized PEG-Tethered Lipid Membrane. Langmuir. 2022 Jun 7;38(22):6959-66.21. Kasashima H, Duran A, Cid-Diaz T, Kudo Y, Diaz-Meco MT, Moscat J. An Orthotopic Implantation Mouse Model of Hepatocellular Carcinoma with Underlying Liver Steatosis. STAR Protocols. 2020 Dec;l(3):100185.22. Duan X, Wang M, Han X, Ren J, Huang G, Ju S, Zhang Q. Combined use of microwave ablation and cell immunotherapy induces nonspecific immunity of hepatocellular carcinoma model mice. Cell Cycle. 2020 Dec 16;19(24):3595-607.299642387.1 - 40 -

Claims

WHAT IS CLAIMED IS:

1. A method of treating hepatocellular carcinoma in a patient, the method comprising administering a galectin- 1 inhibitor the patient.

2. A method of reducing tumor aggressiveness in a hepatocellular carcinoma patient, the method comprising administering a galectin- 1 inhibitor to the patient.

3. The method of claim 1 or 2, wherein patient has received thermal ablation therapy.

4. The method of any one of claims 1 to 3, wherein the patient is indicated to receive thermal ablation therapy.

5. The method of any one of claims 1 to 4, wherein the patient is determined to have an amount of a galectin- 1 gene product, in cancer cells from the patient, higher than a control.

6. The method of claim 5, wherein the control is an amount of a galectin- 1 gene product in cancer cells from a patient determined to be responsive to a thermal ablation therapy.

7. The method of claim 5, wherein the control is an amount of a galectin- 1 gene product in hepatocellular carcinoma cells from a patient determined to be responsive to a thermal ablation therapy.

8. The method of claim 5, wherein the control comprises an amount of galectin- 1 gene product in non-cancerous cells.

9. The method of claim 8, wherein the non-cancerous cells are liver cells.

10. The method of any one of claims 5 to 9, wherein the amount of the galectin- 1 gene product is determined to be at least two-fold higher than the control.

11. The method of any one of claims 5 to 9, wherein the amount of the galectin- 1 gene product is determined to be at least nine-fold higher than the control.

12. The method of any one of claims 1 to 11, wherein the galectin- 1 inhibitor comprises a nucleic acid capable of reducing a galectin- 1 mRNA gene product.

13. The method of any one of claims 1 to 11, wherein the galectin- 1 inhibitor comprises a galectin- 1 mRNA-targeting siRNA.

14. The method of any one of claims 1 to 11, wherein the galectin- 1 inhibitor comprises a galectin- 1 binding protein.299642387.1 - 41 -15. The method of claim 14, wherein the galectin-1 binding protein comprises a galectin-1 antibody.

16. The method of claim 14, wherein the galectin-1 binding protein comprises a carbohydrate, peptide, peptidomimetic, or analog thereof.

17. The method of any one of claims 1 to 11, wherein the galectin-1 inhibitor comprises a galectin-1 targeting gene therapy vector.

18. The method of any one of claims 1 to 17, wherein the galectin-1 inhibitor is capable of reducing the amount of a galectin- 1 gene product in a cancer cell in the patient.

19. The method of any one of claims 1 to 18, wherein the patient is a human patient.

20. A method of measuring a gene profile in a hepatocellular carcinoma patient, the method comprising measuring an amount of a galectin- 1 gene product in cancer cells from the patient.

21. The method of claim 20, wherein patient has received thermal ablation therapy.

22. The method of claim 20 or 21, wherein the patient is indicated to receive thermal ablation therapy.

23. The method of any one of claims 20 to 22, further comprising comparing the measured amount of the galectin-1 gene product to a control.

24. The method of any one of claims 20 to 23, further comprising providing an ablation therapy to the patient when the amount of the galectin-1 gene product is lower than the control.

25. The method of claim 24, wherein the control is an amount of a galectin-1 gene product in cancer cells from a patient determined to be responsive to a thermal ablation therapy.

26. The method of claim 24, wherein the control is an amount of a galectin-1 gene product in hepatocellular carcinoma cells from a patient determined to be responsive to a thermal ablation therapy.

27. The method of claim 24, wherein the control comprises an amount of galectin-1 gene product in non-cancerous cells.

28. The method of claim 27, wherein the non-cancerous cells are liver cells.

29. The method of claim 27 or 28, wherein the non-cancerous cells are taken from the patient.299642387.1 - 42 -30. The method of any one of claims 20 to 29, further comprising administering a galectin-1 inhibitor to the patient.

31. The method of any one of claims 20 to 30, wherein the patient is a human patient.

32. A method of determining tumor aggressiveness and / or responsiveness to a thermal ablation therapy in a hepatocellular carcinoma patient, the method comprising measuring an amount of a galectin- 1 gene product in cancer cells from the patient.

33. The method of claim 32, wherein patient has received thermal ablation therapy.

34. The method of claim 32 or 33, wherein the patient is indicated to receive thermal ablation therapy.

35. The method of any one of claims 32 to 34, further comprising comparing the measured amount of the galectin-1 gene product to a control.

36. The method of any one of claims 32 to 35, further comprising providing an ablation therapy to the patient when the amount of the galectin-1 gene product is lower than the control.

37. The method of claim 36, wherein the control is an amount of a galectin-1 gene product in cancer cells from a patient determined to be responsive to a thermal ablation therapy.

38. The method of claim 36, wherein the control is an amount of a galectin-1 gene product in hepatocellular carcinoma cells from a patient determined to be responsive to a thermal ablation therapy.

39. The method of claim 36, wherein the control comprises an amount of galectin-1 gene product in non-cancerous cells.

40. The method of claim 39, wherein the non-cancerous cells are liver cells.

41. The method of claim 39 or 40, wherein the non-cancerous cells are taken from the patient.

42. The method of any one of claims 32 to 41, further comprising administering a galectin-1 inhibitor to the patient.

43. The method of any one of claims 32 to 42, wherein the patient is a human patient.299642387.1 - 43 -

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