Reliable hexokinase 3 protein detection in human cell lines and primary tissue
Hexokinases (HKs) catalyse the first and rate limiting step of glycolysis by converting glucose into glucose-6-phosphate (G6P). The 100kDa enzymes HK1-3 are thought to have arisen from a common 50kDa precursor by gene duplication and tandem ligation. The fourth 50kDa isoform HK4 with its high degree in sequence similarity supports this hypothesis. HK1 and 3 show catalytic activity at the C-terminal half and glucose binding properties at the N-terminal half, whereas in HK2 both the N-terminal and C-terminal halves are catalytically active. All HKs show a cytoplasmic localization whereas HK1 and HK2 can bind to the outer mitochondrial membrane via a 21 hydrophobic amino acid sequence at their N-terminus. HK3 however, does not contain the conserved mitochondrial binding region. On the subcellular level the HK2 isoform has also been shown to localize to the nucleus and HK3 may have a perinuclear localization. Although the primary function of HKs is linked to glycolysis, these enzymes have been shown to play additional regulatory roles within cells. Depending on factors such as cell type, subcellular localization, and the cell’s metabolic state, different isoforms of HK are involved in diverse processes such as autophagy, cell death, DNA damage, and reactive oxygen species (ROS) production. For instance, mitochondrial HK1 and HK2 inhibit apoptosis by selectively translocating proapoptotic Bcl-2 family members such as tBid, Bax, and Bak away from the mitochondria. Nuclear HK2, on the other hand, maintains stem cell fate, modulates chromatin accessibility, and enhances the DNA damage response. Among the HK family, HK3 has gained particular attention for its pro-survival role in hematological malignancies, specifically acute promyelocytic leukemia (APL) and acute myeloid leukemia (AML). Notably, HK3 is a transcriptional target of PU.1 7 a critical regulator of myeloid differentiation, highlighting its potential importance in both AML and myeloid cells in general. However, research into HK3 has been hindered by the lack of a highly specific antibody. This has made it difficult to study HK3’s expression and function using traditional methods such as Western blotting. As a result, researchers have had to rely on alternative techniques, such as PCR or endogenous tagging, which are more cumbersome and semi-artificial techniques. Moreover, the absence of a specific antibody may have contributed to some preliminary misinterpretations or incomplete conclusions regarding HK3’s role, hindering a more comprehensive understanding of its functions. 

Cell lines, culture and treatment conditions HEK 293T cells were maintained in DMEM supplemented with 5% fetal bovine serum (FBS), 1% HEPES and were kept in 7.5% CO 2 -92.5 % air humified atmosphere at 37°C. The human AML cell lines HL60, THP1 and OCI-AML2 were maintained in RPMI-1640 with 10% FBS in 5% CO 2 -95% air humified atmosphere at 37°C. HL60 cells were differentiated into neutrophils with 1 µM all-trans retinoic acid (ATRA) (DMSO, Merck KGaA Darmstadt, Germany) over 4 days. Macrophage differentiation of HL60 and THP1 cells was induced with 1 nM phorbol-12-myristate-13-acetate (PMA) (DMSO) for 48 h or 65 nM PMA for 24 h respectively. The solid cancer cell lines A549 and HCT116 were maintained in DMEM/F12 with 10%FBS and DMEM with 10%FBS and 1% L-Glutamine, respectively. Cells were kept in 5% CO 2 -95% air humified atmosphere at 37°C. Generation of knockout cell lines HK3 knockout cell lines were generated using the lentiCRISPRv2 vector containing the Cas9 endonuclease gene, gRNA (HK3: GGATGCTGCCTACATACGTG) and a puromycin selection marker. Lentiviral vectors for CRISPR knockouts were generated by transient transfection of the corresponding plasmid and third-generation packaging plasmids pMD2.G (VSV-G), pMDLg/pRRE (gag and pol), and pRSV-Rev (rev) into HEK 293 T cells. Briefly, cells were transfected using calcium phosphate, and the supernatant was collected 72 h post transfection. The media was filtered through a 0.45 μm nitrocellulose membrane. About 4x10 5 THP1 cells were incubated with 400 μL virus and 8 μg/mL polybrene; after 4 h a second transduction was performed. After 2 days cells were selected with 1.5 μg/mL puromycin for 4 days followed by 3 days of 0.5 μg/mL. Following selection cells were sorted using HANA Cell Sorter and Single Cell Dispenser (Biotechne, Minneapolis, MN, USA) and single cells grown in the 96-well format. Endogenous HiBit-tagging of hexokinase 3 Endogenous tagging of HK3 with the HiBiT sequence was performed following the published protocol using the Neon Electroporation Kit (Invitrogen, Carlsbad, CA, USA). Alt-R HiFi Cas9, ssODN, sgRNA, and HDR enhancer were obtained from Integrated DNA Technologies (IDT, Coralville, IA, USA). Briefly, the RNP complex was assembled by incubating sgRNA with Alt-R HiFi Cas9 in a 2.5:1 ratio in Buffer R for 15 min at room temperature (RT). The ssODN was then added to the RNP mix, which was subsequently incubated at 4°C. Concurrently, cRPMI was preincubated with HDR enhancer (1 µM), while HL60 cells were washed with PBS and resuspended at a concentration of 2×10 7 cells/mL in Buffer R. The RNP and ssODN mix was then added to the cells, which were electroporated using a 10 µL tip with the following settings: 1 pulse at 1700 V for 20 ms. After 24 h, the HDR-containing medium was replaced with fresh medium containing 20% FBS. A HiBiT lytic assay was performed 96 h post-electroporation to detect luminescence. HiBiT lytic detection Luminescent detection of HiBit was performed using Nano-Glo ® HiBit Lytic detection System according to manufacturer’s instructions (Promega, Madison, WI, USA). Briefly, adding the cells (1:1) to the reaction buffer (prepared by adding 1:50 Substrate and 1:100 LgBit in assay buffer) in 96 well flat white plates and read after 10 min using Tecan Reader with 1000 ms integration time. The values were normalized to cell number using CellTiter-Glo ® luminescent cell viability assay (Promega). Transient transfection HEK 293T cells were transiently transfected with lentiviral plasmids containing Flag-HK1, Flag-HK2 or Flag-HK3, using the calcium-phosphate method. In total 10 µg of plasmid was used and after the transfection cells were cultured in 5% CO 2 -95% humified atmosphere at 37°C for 48 h. Western blotting Whole cell extracts were prepared using RIPA buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X lysis buffer) with addition of protease inhibitor (Roche, Basel, Switzerland). Protein concentration was measured by Bradford Assay (BioRad, Hercules, CA, USA) and 0.4-25 µg total protein was loaded onto 12% TGX stain free, fast cast acrylamide gels (BioRad). Blots were incubated with the primary antibodies in TBS 0.1% Tween-20/ 5% non-fat milk or 5% BSA overnight at 4°C plus 2 h at RT, then incubated with HRP coupled secondary anti-rabbit or anti-mouse antibody at 1:3000-1:10000 for 1-3 h at RT. For re-incubation membranes were stripped using 1.5% Glycine (w/v), 0.1% SDS (w/v), 1% Tween-20 (v/v) in dH 2 O, pH 2.2. Successful stripping was verified by incubation with the according secondary antibody and visualized with ECL solution (BioRad). Primary antibodies used are listed in . Uncropped Western blots and stripped membranes are shown in the supplement. Embedding of cells for immunohistochemical Labeling Cells were pelleted by centrifugation before 3-4 drops of plasma (Blood Transfusion Service, Bern, Switzerland) and 1-2 drops of thrombin (Diagnotec, Liestal, Switzerland) were added to initiate coagulation. After 2 min of incubation, the cell pellet was transferred into an embedding cassette. The sample was then fixed in 4% paraformaldehyde overnight at RT. After fixation, the cells were embedded in paraffin following standard procedures in the routine pathology laboratory. Immunohistochemical Labeling Before immunohistochemical Labeling paraffin-embedded samples were pre-processed, which included de-paraffination, rehydration, and antigen retrieval. IHC Labeling was performed using an automated immunostainer (Bond RX; Leica Biosystems, Heerbrugg, Switzerland). Antibodies used were HK3 (Merck KGaA; HPA056743 dilution 1:2000) and the CD68 antibody (Dako Omnis, Agilent, Santa Clara, CA, USA; clone PG-M1; dilution 1:200). Visualization was performed using the Bond Polymer Refine Detection kit (Leica Biosystems) as instructed by the manufacturer. The ratio of HK3/CD68 positive cells was determined by counting the positive cells in an area of 2 mm of sequential cuts. 

HEK 293T cells were maintained in DMEM supplemented with 5% fetal bovine serum (FBS), 1% HEPES and were kept in 7.5% CO 2 -92.5 % air humified atmosphere at 37°C. The human AML cell lines HL60, THP1 and OCI-AML2 were maintained in RPMI-1640 with 10% FBS in 5% CO 2 -95% air humified atmosphere at 37°C. HL60 cells were differentiated into neutrophils with 1 µM all-trans retinoic acid (ATRA) (DMSO, Merck KGaA Darmstadt, Germany) over 4 days. Macrophage differentiation of HL60 and THP1 cells was induced with 1 nM phorbol-12-myristate-13-acetate (PMA) (DMSO) for 48 h or 65 nM PMA for 24 h respectively. The solid cancer cell lines A549 and HCT116 were maintained in DMEM/F12 with 10%FBS and DMEM with 10%FBS and 1% L-Glutamine, respectively. Cells were kept in 5% CO 2 -95% air humified atmosphere at 37°C. Generation of knockout cell lines HK3 knockout cell lines were generated using the lentiCRISPRv2 vector containing the Cas9 endonuclease gene, gRNA (HK3: GGATGCTGCCTACATACGTG) and a puromycin selection marker. Lentiviral vectors for CRISPR knockouts were generated by transient transfection of the corresponding plasmid and third-generation packaging plasmids pMD2.G (VSV-G), pMDLg/pRRE (gag and pol), and pRSV-Rev (rev) into HEK 293 T cells. Briefly, cells were transfected using calcium phosphate, and the supernatant was collected 72 h post transfection. The media was filtered through a 0.45 μm nitrocellulose membrane. About 4x10 5 THP1 cells were incubated with 400 μL virus and 8 μg/mL polybrene; after 4 h a second transduction was performed. After 2 days cells were selected with 1.5 μg/mL puromycin for 4 days followed by 3 days of 0.5 μg/mL. Following selection cells were sorted using HANA Cell Sorter and Single Cell Dispenser (Biotechne, Minneapolis, MN, USA) and single cells grown in the 96-well format. Endogenous HiBit-tagging of hexokinase 3 Endogenous tagging of HK3 with the HiBiT sequence was performed following the published protocol using the Neon Electroporation Kit (Invitrogen, Carlsbad, CA, USA). Alt-R HiFi Cas9, ssODN, sgRNA, and HDR enhancer were obtained from Integrated DNA Technologies (IDT, Coralville, IA, USA). Briefly, the RNP complex was assembled by incubating sgRNA with Alt-R HiFi Cas9 in a 2.5:1 ratio in Buffer R for 15 min at room temperature (RT). The ssODN was then added to the RNP mix, which was subsequently incubated at 4°C. Concurrently, cRPMI was preincubated with HDR enhancer (1 µM), while HL60 cells were washed with PBS and resuspended at a concentration of 2×10 7 cells/mL in Buffer R. The RNP and ssODN mix was then added to the cells, which were electroporated using a 10 µL tip with the following settings: 1 pulse at 1700 V for 20 ms. After 24 h, the HDR-containing medium was replaced with fresh medium containing 20% FBS. A HiBiT lytic assay was performed 96 h post-electroporation to detect luminescence. 

HK3 knockout cell lines were generated using the lentiCRISPRv2 vector containing the Cas9 endonuclease gene, gRNA (HK3: GGATGCTGCCTACATACGTG) and a puromycin selection marker. Lentiviral vectors for CRISPR knockouts were generated by transient transfection of the corresponding plasmid and third-generation packaging plasmids pMD2.G (VSV-G), pMDLg/pRRE (gag and pol), and pRSV-Rev (rev) into HEK 293 T cells. Briefly, cells were transfected using calcium phosphate, and the supernatant was collected 72 h post transfection. The media was filtered through a 0.45 μm nitrocellulose membrane. About 4x10 5 THP1 cells were incubated with 400 μL virus and 8 μg/mL polybrene; after 4 h a second transduction was performed. After 2 days cells were selected with 1.5 μg/mL puromycin for 4 days followed by 3 days of 0.5 μg/mL. Following selection cells were sorted using HANA Cell Sorter and Single Cell Dispenser (Biotechne, Minneapolis, MN, USA) and single cells grown in the 96-well format. 

Endogenous tagging of HK3 with the HiBiT sequence was performed following the published protocol using the Neon Electroporation Kit (Invitrogen, Carlsbad, CA, USA). Alt-R HiFi Cas9, ssODN, sgRNA, and HDR enhancer were obtained from Integrated DNA Technologies (IDT, Coralville, IA, USA). Briefly, the RNP complex was assembled by incubating sgRNA with Alt-R HiFi Cas9 in a 2.5:1 ratio in Buffer R for 15 min at room temperature (RT). The ssODN was then added to the RNP mix, which was subsequently incubated at 4°C. Concurrently, cRPMI was preincubated with HDR enhancer (1 µM), while HL60 cells were washed with PBS and resuspended at a concentration of 2×10 7 cells/mL in Buffer R. The RNP and ssODN mix was then added to the cells, which were electroporated using a 10 µL tip with the following settings: 1 pulse at 1700 V for 20 ms. After 24 h, the HDR-containing medium was replaced with fresh medium containing 20% FBS. A HiBiT lytic assay was performed 96 h post-electroporation to detect luminescence. 

Luminescent detection of HiBit was performed using Nano-Glo ® HiBit Lytic detection System according to manufacturer’s instructions (Promega, Madison, WI, USA). Briefly, adding the cells (1:1) to the reaction buffer (prepared by adding 1:50 Substrate and 1:100 LgBit in assay buffer) in 96 well flat white plates and read after 10 min using Tecan Reader with 1000 ms integration time. The values were normalized to cell number using CellTiter-Glo ® luminescent cell viability assay (Promega). 

HEK 293T cells were transiently transfected with lentiviral plasmids containing Flag-HK1, Flag-HK2 or Flag-HK3, using the calcium-phosphate method. In total 10 µg of plasmid was used and after the transfection cells were cultured in 5% CO 2 -95% humified atmosphere at 37°C for 48 h. 

Whole cell extracts were prepared using RIPA buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X lysis buffer) with addition of protease inhibitor (Roche, Basel, Switzerland). Protein concentration was measured by Bradford Assay (BioRad, Hercules, CA, USA) and 0.4-25 µg total protein was loaded onto 12% TGX stain free, fast cast acrylamide gels (BioRad). Blots were incubated with the primary antibodies in TBS 0.1% Tween-20/ 5% non-fat milk or 5% BSA overnight at 4°C plus 2 h at RT, then incubated with HRP coupled secondary anti-rabbit or anti-mouse antibody at 1:3000-1:10000 for 1-3 h at RT. For re-incubation membranes were stripped using 1.5% Glycine (w/v), 0.1% SDS (w/v), 1% Tween-20 (v/v) in dH 2 O, pH 2.2. Successful stripping was verified by incubation with the according secondary antibody and visualized with ECL solution (BioRad). Primary antibodies used are listed in . Uncropped Western blots and stripped membranes are shown in the supplement. 

Cells were pelleted by centrifugation before 3-4 drops of plasma (Blood Transfusion Service, Bern, Switzerland) and 1-2 drops of thrombin (Diagnotec, Liestal, Switzerland) were added to initiate coagulation. After 2 min of incubation, the cell pellet was transferred into an embedding cassette. The sample was then fixed in 4% paraformaldehyde overnight at RT. After fixation, the cells were embedded in paraffin following standard procedures in the routine pathology laboratory. 

Before immunohistochemical Labeling paraffin-embedded samples were pre-processed, which included de-paraffination, rehydration, and antigen retrieval. IHC Labeling was performed using an automated immunostainer (Bond RX; Leica Biosystems, Heerbrugg, Switzerland). Antibodies used were HK3 (Merck KGaA; HPA056743 dilution 1:2000) and the CD68 antibody (Dako Omnis, Agilent, Santa Clara, CA, USA; clone PG-M1; dilution 1:200). Visualization was performed using the Bond Polymer Refine Detection kit (Leica Biosystems) as instructed by the manufacturer. The ratio of HK3/CD68 positive cells was determined by counting the positive cells in an area of 2 mm of sequential cuts. 

Hexokinase 3: a ‘white blood cell hexokinase’ with expression predominantly associated with myeloid cells Upon analyzing the publicly available datasets on the human protein atlas (HPA), an enrichment of HK3 gene expression in bone marrow and lymphoid tissues becomes evident ( ). A closer examination at the single-cell level reveals that the highest HK3 mRNA expression occurs in phagocytic cells, including macrophages, monocytes, and tissue-resident macrophages like Hofbauer cells in the placenta and Kupffer cells in the liver ( ). We then focused on HK3 mRNA expression across various immune cell types and found it to be predominantly associated with cells of myeloid origin ( ). This observation prompted us to explore HK3 expression during myeloid differentiation, where we noted a 2-fold increase in HK3 transcript levels as differentiation progressed ( ). If we then examined HK3 levels in cancer cell lines published on the HPA, it becomes apparent that HK3 mRNA is confined to myeloid leukemia cell lines, with no transcripts detected in solid cancer cell lines ( ). Testing several commercially available HK3 antibodies for their specificity by Western blotting The HK family consists of isoenzymes that, despite being located on different chromosomes, exhibit up to 70% sequence and structural identity. The three HKs, HK1, HK2 and HK3 are comprised of two highly analogous domains: an N-terminal domain and a C-terminal domain. HK3, however, lacks significant similarity to other HK isoforms at its N-terminus due to the absence of the mitochondrial binding domain. In terms of the small and large sub-domains of HKs, the shared sequence identity of HK3 with other isoforms ranges between 44% and 54%. At the C-terminal region, the sequence similarity among the HK isoforms is higher, ranging from 61% to 70% ( ). Due to the pronounced sequence similarity among HKs, identifying a truly specific antibody for HK3 is challenging. We evaluated several commercially available antibodies for HK3, some of which have been used in other studies exploring the role of HK3 in solid cancers. The immunogen sequence used to produce the respective antibodies are depicted in . To assess specificity, these antibodies were tested on 293T cells, which have no detectable basal expression of HK3 mRNA according to the HPA ( ). The cells were transiently transfected to overexpress Flag-HK1, Flag-HK2, or Flag-HK3, and serial dilutions of protein were loaded to evaluate antibody sensitivity ( ). We used THP1, an AML cell line with moderate HK3 expression according to the HPA, as a positive control. THP1 cells depleted of HK3 using CRISPRCas9 technology (validated by TIDE analysis; Supplementary Figure S1 A,B ) served as negative control. Our results indicate that most tested antibodies for HK3 also cross-react with HK1 and HK2, often detecting them more effectively than HK3 ( ). Lastly, we verified that HK1 and HK2 are indeed expressed at endogenous levels in THP1 HK3 knockout (KO) cells as well as in 293T cells that were used for transient transfections of the different HK expression plasmids ( ). The notable exception not showing cross-reactivity was the Prestige Antibody (HPA056743), which specifically recognized HK3. This specificity can be attributed to its recognition site at the N-terminus, where there is no significant sequence similarity with HK1 or HK2 ( ). Expression of HK3 is absent in solid cancer cells but induced upon myeloid differentiation According to mRNA transcript data from the HPA, solid cancer cell lines do not express HK3 mRNA ( ). To validate this, we determined HK3 protein levels in the lung adenocarcinoma cell line (A549) and the colorectal cancer cell line (HCT116). By comparing the results from different antibodies, we observed that only the HK3 specific Prestige antibody (HPA056743) confirmed the absence of the HK3 protein in these cell lines. In contrast, the other two antibodies, Thermo Fisher (PA5-29304) and Proteintech (67803-1-IG), detected non-specific bands corresponding very likely to other HKs (Figure 3A; Supplementary Figure S2A ). We further investigated whether HK3 expression levels change during myeloid differentiation. Therefore, we utilized the AML cell line HL60, which can be induced to differentiate into neutrophils in response to ATRA or into macrophages using PMA. We also included the THP1 AML cell line, which differentiates into macrophages upon PMA treatment. In both cell lines, HK3 expression levels increased after differentiation (Figure 3A; Supplementary Figure S2B). In addition to the THP1 HK3 KO cells we also used HL60 HK3 KO cells (confirmed by MS analysis ) as negative controls. To confirm these findings, we employed a highly sensitive HK3-HiBiT-endogenous tagging system in the HL60 cell line. Following endogenous tagging of HK3 with the HiBiT tag, we assessed HK3 expression using a lytic luminescence assay, which further demonstrated the elevated expression of HK3 upon neutrophil and macrophage differentiation in HL60 cells ( ). Anti-HK3 antibody from Prestige antibodies specifically stains macrophages in tissue IHC Immunohistochemistry (IHC) is a valuable tool in translational research, particularly for detecting protein expression in patient samples. To ensure the Prestige antibody’s effectiveness and specificity in IHC, we validated it using two AML cell lines (OCI AML2 and THP1) known to express HK3. Both parental and HK3 knockout cell lines were labeled and the results showed HK3 detection in the parental cell lines, with no expression in the KO variants ( ). Next, we sought to identify the cell types expressing HK3 in lung and colon tissues. To achieve this, we analyzed published single-cell RNA sequencing datasets, including the Human Lung Cell Atlas (HLCA) and the Human CD Atlas on the Single Cell Portal, alongside the HPA dataset. In the lung, both HLCA and HPA datasets demonstrated that HK3 expression is restricted to macrophage and monocyte clusters ( ). These findings are consistent with our IHC labeling, which revealed robust HK3 labeling in large mononuclear cells characterized by round, oval, or kidney-shaped nuclei and abundant granular cytoplasm-a morphology consistent with macrophages. Notably, epithelial and alveolar cells, as well as all other cell types, lacked HK3 labeling ( ). In the colon, HK3 expression was similarly confined to macrophage and monocyte immune subsets ( ). Among all cell types in the colon, the highest enrichment of HK3 was observed in macrophages and neutrophils ( ). This was corroborated by CD68 labeling of macrophages in the same colon tissues, which revealed a similar distribution pattern of HK3 expression in these cells ( ). Sequential sections of normal colon tissue demonstrated a ratio of HK3-positive to CD68-positive cells of 132:490. These results align with single-cell RNA sequencing data from colon immune tissues, which reported that only a subset of macrophages exhibits HK3 positivity. Together, these findings highlight the selective expression of HK3 in macrophages and monocytes across lung and colon tissues, emphasizing its potential role in immune cell-specific functions. 

Upon analyzing the publicly available datasets on the human protein atlas (HPA), an enrichment of HK3 gene expression in bone marrow and lymphoid tissues becomes evident ( ). A closer examination at the single-cell level reveals that the highest HK3 mRNA expression occurs in phagocytic cells, including macrophages, monocytes, and tissue-resident macrophages like Hofbauer cells in the placenta and Kupffer cells in the liver ( ). We then focused on HK3 mRNA expression across various immune cell types and found it to be predominantly associated with cells of myeloid origin ( ). This observation prompted us to explore HK3 expression during myeloid differentiation, where we noted a 2-fold increase in HK3 transcript levels as differentiation progressed ( ). If we then examined HK3 levels in cancer cell lines published on the HPA, it becomes apparent that HK3 mRNA is confined to myeloid leukemia cell lines, with no transcripts detected in solid cancer cell lines ( ). 

The HK family consists of isoenzymes that, despite being located on different chromosomes, exhibit up to 70% sequence and structural identity. The three HKs, HK1, HK2 and HK3 are comprised of two highly analogous domains: an N-terminal domain and a C-terminal domain. HK3, however, lacks significant similarity to other HK isoforms at its N-terminus due to the absence of the mitochondrial binding domain. In terms of the small and large sub-domains of HKs, the shared sequence identity of HK3 with other isoforms ranges between 44% and 54%. At the C-terminal region, the sequence similarity among the HK isoforms is higher, ranging from 61% to 70% ( ). Due to the pronounced sequence similarity among HKs, identifying a truly specific antibody for HK3 is challenging. We evaluated several commercially available antibodies for HK3, some of which have been used in other studies exploring the role of HK3 in solid cancers. The immunogen sequence used to produce the respective antibodies are depicted in . To assess specificity, these antibodies were tested on 293T cells, which have no detectable basal expression of HK3 mRNA according to the HPA ( ). The cells were transiently transfected to overexpress Flag-HK1, Flag-HK2, or Flag-HK3, and serial dilutions of protein were loaded to evaluate antibody sensitivity ( ). We used THP1, an AML cell line with moderate HK3 expression according to the HPA, as a positive control. THP1 cells depleted of HK3 using CRISPRCas9 technology (validated by TIDE analysis; Supplementary Figure S1 A,B ) served as negative control. Our results indicate that most tested antibodies for HK3 also cross-react with HK1 and HK2, often detecting them more effectively than HK3 ( ). Lastly, we verified that HK1 and HK2 are indeed expressed at endogenous levels in THP1 HK3 knockout (KO) cells as well as in 293T cells that were used for transient transfections of the different HK expression plasmids ( ). The notable exception not showing cross-reactivity was the Prestige Antibody (HPA056743), which specifically recognized HK3. This specificity can be attributed to its recognition site at the N-terminus, where there is no significant sequence similarity with HK1 or HK2 ( ). 

According to mRNA transcript data from the HPA, solid cancer cell lines do not express HK3 mRNA ( ). To validate this, we determined HK3 protein levels in the lung adenocarcinoma cell line (A549) and the colorectal cancer cell line (HCT116). By comparing the results from different antibodies, we observed that only the HK3 specific Prestige antibody (HPA056743) confirmed the absence of the HK3 protein in these cell lines. In contrast, the other two antibodies, Thermo Fisher (PA5-29304) and Proteintech (67803-1-IG), detected non-specific bands corresponding very likely to other HKs (Figure 3A; Supplementary Figure S2A ). We further investigated whether HK3 expression levels change during myeloid differentiation. Therefore, we utilized the AML cell line HL60, which can be induced to differentiate into neutrophils in response to ATRA or into macrophages using PMA. We also included the THP1 AML cell line, which differentiates into macrophages upon PMA treatment. In both cell lines, HK3 expression levels increased after differentiation (Figure 3A; Supplementary Figure S2B). In addition to the THP1 HK3 KO cells we also used HL60 HK3 KO cells (confirmed by MS analysis ) as negative controls. To confirm these findings, we employed a highly sensitive HK3-HiBiT-endogenous tagging system in the HL60 cell line. Following endogenous tagging of HK3 with the HiBiT tag, we assessed HK3 expression using a lytic luminescence assay, which further demonstrated the elevated expression of HK3 upon neutrophil and macrophage differentiation in HL60 cells ( ). 

Immunohistochemistry (IHC) is a valuable tool in translational research, particularly for detecting protein expression in patient samples. To ensure the Prestige antibody’s effectiveness and specificity in IHC, we validated it using two AML cell lines (OCI AML2 and THP1) known to express HK3. Both parental and HK3 knockout cell lines were labeled and the results showed HK3 detection in the parental cell lines, with no expression in the KO variants ( ). Next, we sought to identify the cell types expressing HK3 in lung and colon tissues. To achieve this, we analyzed published single-cell RNA sequencing datasets, including the Human Lung Cell Atlas (HLCA) and the Human CD Atlas on the Single Cell Portal, alongside the HPA dataset. In the lung, both HLCA and HPA datasets demonstrated that HK3 expression is restricted to macrophage and monocyte clusters ( ). These findings are consistent with our IHC labeling, which revealed robust HK3 labeling in large mononuclear cells characterized by round, oval, or kidney-shaped nuclei and abundant granular cytoplasm-a morphology consistent with macrophages. Notably, epithelial and alveolar cells, as well as all other cell types, lacked HK3 labeling ( ). In the colon, HK3 expression was similarly confined to macrophage and monocyte immune subsets ( ). Among all cell types in the colon, the highest enrichment of HK3 was observed in macrophages and neutrophils ( ). This was corroborated by CD68 labeling of macrophages in the same colon tissues, which revealed a similar distribution pattern of HK3 expression in these cells ( ). Sequential sections of normal colon tissue demonstrated a ratio of HK3-positive to CD68-positive cells of 132:490. These results align with single-cell RNA sequencing data from colon immune tissues, which reported that only a subset of macrophages exhibits HK3 positivity. Together, these findings highlight the selective expression of HK3 in macrophages and monocytes across lung and colon tissues, emphasizing its potential role in immune cell-specific functions. 

The structurally and on a sequence level very similar HK isoforms 1-3 show distinct tissue expression. HK1 is ubiquitously expressed, whereas HK2 is limited to insulin sensitive tissue such as skeletal muscle and adipose tissue. Lately researchers have focused on the role of HK3 in solid cancers and concluded it to have an effect on glycolysis in intestinal microbial-induced metastasis of colorectal cancer (CRC), that it’s overexpression promotes orthotopic tumor growth and that HK3 supported malignant biologic behaviour in renal cell carcinoma cell lines, and it’s inhibition promotes invasion of hepatocellular carcinoma cells (HCC). In our study and in publicly available databases, however, we found that HK3 is almost exclusively expressed in cells of myeloid origin and that no HK3 mRNA could be detected in solid cancer cell lines under basal conditions. Our previously published paper, Seiler et al . demonstrates an upregulation of HK3 during myeloid differentiation, consistent with the analysis of publicly available datasets and the experimental outcomes presented in this paper. These findings confirm increased HK3 levels in more differentiated myeloid cells. Despite this tissue specific expression, the relatively high degree of sequence similarity between the HK isoforms can lead to cross reactivity of antibodies and potentially inaccurate interpretations. In our hands only 1/4 commercially available antibodies tested showed specific binding to HK3 on Western blotting and IHC. Antibody validation and characterization of specificity is not a new concern; this issue has been recognized and discussed extensively with the challenges nicely laid out here. Several reports, including those from our group , and others have highlighted the challenges posed by poorly validated antibodies. The so-called “antibody characterization crisis” has been thoroughly reviewed. Over the years, various solutions have been proposed to address these issues, offering valuable suggestions for both companies and researchers. We believe that reporting problems with antibody specificities are critical for the scientific community for correct interpretation of results. Our work with HK3 serves as a clear example: many studies have drawn misleading conclusions about HK3 expression that may be due to antibody cross-reactivity. While these findings are not entirely erroneous, the data become clearer when combined with RNA-seq data from bulk sequencing. Our interpretation is that the observed HK3 expression is largely attributable to immune cells present in the tumor microenvironment of solid cancers as we could only detect HK3 expression in the macrophages of tissues and it was nicely demonstrated by Liang et al . naming it a ‘potential immune-related biomarker’. 23 The precise role of HK3 in these contexts, however, remains an open question that requires further investigation. Our results have shown that HK3 expression is predominantly found in myeloid cells, though it can be induced even in cells with low basal levels of HK3 under certain stimuli. Whether this holds true in non-hematopoietic cells is still unresolved. With the development of proper tools and more rigorous validation methods, we hope to have laid the groundwork for future studies on HK3, enabling researchers to explore its role with greater accuracy and confidence. 

