Tumor Vessel Normalization via PFKFB3 Inhibition Alleviates Hypoxia and Increases Tumor Necrosis in Rectal Cancer upon Radiotherapy

Abstract Treatment of patients with locally advanced rectal cancer (RC) is based on neoadjuvant chemoradiotherapy followed by surgery. In order to reduce the development of therapy resistance, it is necessary to further improve previous treatment approaches. Recent in vivo experimental studies suggested that the reduction of tumor hypoxia by tumor vessel normalization (TVN), through the inhibition of the glycolytic activator PFKFB3, could significantly improve tumor response to therapy. We have evaluated in vitro and in vivo the effects of the PFKFB3 inhibitor 2E-3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) on cell survival, clonogenicity, migration, invasion, and metabolism using colorectal cancer cells, patient-derived tumor organoid (PDO), and xenograft (PDX). 3PO treatment of colorectal cancer cells increased radiation-induced cell death and reduced cancer cell invasion. Moreover, gene set enrichment analysis shows that 3PO is able to alter the metabolic status of PDOs toward oxidative phosphorylation. Additionally, in vivo neoadjuvant treatment with 3PO induced TVN, alleviated tumor hypoxia, and increased tumor necrosis. Our results support PFKFB3 inhibition as a possible future neoadjuvant addition for patients with RC. Significance: Novel therapies to better treat colorectal cancer are necessary to improve patient outcomes. Therefore, in this study, we evaluated the combination of a metabolic inhibitor (3PO) and standard radiotherapy in different experimental settings. We have observed that the addition of 3PO increased radiation effects, ultimately improving tumor cell response to therapy.


Introduction
Colorectal cancer (CRC) is the third most common cancer diagnosed worldwide and the second leading cause of cancer death (1).When further stratified, the worldwide mortality of patients with colon and rectal cancer (RC) accounts to approximately 500,000 and 310,000 cases yearly, respectively (1).Treatment of patients with locally advanced RC is based on neoadjuvant chemoradiotherapy (CRT) followed by conventional surgery.
Patients' response to preoperative radiotherapy (RT) is heterogeneous and ranges from resistance and disease progression despite intensive treatment to complete regression of the tumor (clinical complete response).The extent of tumor regression correlates with the long-term clinical outcome (2,3).There are several studies assessing the benefits of including patients who respond fully to CRT in watch-and-wait strategies showing a striking rate of organ preservation warranting further prospective validation (4,5).However, a relevant number of patients develop tumor regrowth, and this concept should be further evaluated within prospective clinical trials.It is already well known that the content of oxygen within the tumor tissue plays a crucial role during RT (6,7).Radiation-resistant cells are found particularly in hypoxic regions; therefore, it is not surprising that solid tumors with high oxygenation show a significantly better response to RT than those that are com-xCELLigence assay xCELLigence assay was performed as previously described (18).Shortly, by measuring the impedance of bottom coated plates with microelectrodes, the xCELLigence software provides a method to analyze cell proliferation, adhesion, morphology, and death in real time.Changes in cell size, cell number, and cell adhesion result in changes of impedance, which are reflected as cell index (CI).Every condition was tested in triplicates, and the assay was performed three times.Conditions were compared by fitting a saturation curve in logarithm and predicting the 80-hour time point which was then compared using a t test.
Every condition was tested in triplicates, and the assay was performed three times.

Invasion assay
The invasion assay was performed using a Boyden chamber, coated with Cultrex Basement Membrane Extract, PathClear (R&D Systems, MN, USA), as described previously (19). 1 � 10 5 cells were seeded into the upper well of

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Cancer Res Commun; 4(8) August 2024 2009 the Boyden chamber in a 1,000 µL medium.3PO was added into both sides of the chamber.For the invasion assay in combination with irradiation, CRC cells were irradiated in 75-mL cell culture flask using RS 225 X-ray irradiation system (200 kV, 15 mA, 0.5 mm Cu filter, Gulmay Medical System, Xstrahl Ltd., Camberley, Surrey, UK) with a single dose of 6 Gy.Next, irradiated cells were seeded into the Boyden chamber.After 96 hours of incubation at 37 °C and 5% CO 2 , the medium from the upper chamber was removed, and the content of the lower part, containing the invaded cells (both floating and adherent cells), was collected.Cells were centrifuged at 500 g for 5 minutes, stained with 0.4% Trypan Blue (Carl Roth, Karlsruhe, Germany) and counted manually using a Neubauer-improved chamber (Paul Marienfeld GmbH, Lauda-Königshofen, Germany).Every condition was tested in triplicates, and the assay was performed three times.

Migration assay
Migration assay was performed as previously described (18), with cells previously incubated in 0.5 µmol/L mitomycin C for 4 hours.Silicone inserts (ibidi Integrated Biodiagnostics, Germany) were removed when cell confluency was reached (100%).Migration assay in combination with irradiation was performed using RS 225 X-ray irradiation system (200 kV, 15 mA, 0.5mm copper filter, Gulmay Medical System, Xstrahl Ltd., Camberley, Surrey, UK), 4 hours after cells were exposed to 3PO with a single dose of 6 Gy.
Analyses were performed using ImageJ 1.43u software (National Institutes of Health, Bethesda, Maryland, USA.1997-2016) and GraphPad Prism 9 (GraphPad Software, Inc., La Jolla, CA, RRID:SCR_002798).Migration was calculated with the following formula: migration (%) ¼ A0/Ax.Changes of the area of the gap (Ax) were set in relation to the area of the gap at a time of 0 hour (A0).The resulting percentage describes the migration ability of the cells.

Spheroid formation assay
For spheroid formation, 5 � 10 5 HUVEC cells were mixed with 10-mL Endothelial Cell Growth Medium 2 (ECGM2, PromoCell, Heidelberg, Germany, RRID:SCR_023579) supplemented with 2% v/w sterile methylcellulose (Sigma-Aldrich, St. Louis, USA).Tthe cell suspension (25 µL) was turned upside down (hanging drops) for overnight incubation (approx.16 hours).The next day, spheroids were gently washed and collected into a 50-mL conical tube with approximately 10 mL of PBS (Gibco) supplemented with 10% FBS (Biochrom).After 5 minutes of centrifugation at 300 g at RT with no brakes, ECGM2 medium (PromoCell) supplemented with 5 mg/mL fibrinogen (Sigma-Aldrich) was gently overlayed on the pellet and mixed.For solidification, 0.6 U (approx. 6 µL/mL) thrombin (Sigma-Aldrich) was added and again gently mixed.Approximately 500 µL of the mixture-containing spheroids was platted into 24-well plates (SARSTEDT, Hildesheim, Germany).Spheroids were cultured for 24 hours, with medium containing DMSO (control) or with 3PO.After 24 hours, cultures' images were captured, and in vitro angiogenesis was quantified digitally by measuring the length and number of the sprouts (calculated as cumulative sprout length).
For the treatment of well-stablished spheroid sprouts, we have allowed sprouts to form for 24 hours and then add 3PO in different concentrations into the medium for another 24 hours.Images were analyzed using the ImageJ 1.53a software (National Institutes of Health, Bethesda, Maryland, USA.1997-2016).At least five spheroids per each independent experiment were analyzed.

RT-PCR
RNA extraction and real-time PCR analysis were performed as recently described (18).

Patient-derived organoids and Western blot analysis
Fresh tissue samples used in this study were provided by the University Medical Center Göttingen (UMG), Germany, after surgical tumor resection (ethical approval UMG Antragsnr.25/3/17, in line with the ethical principles of the Declaration of Helsinki).
Western blot analysis was performed as previously described (18), with

Histology and immunostainings
The methods for histology and immunostainings have been described previously (14).Immunostainings were done with the following antibodies: CD-

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed with paraffin sections, using the DeadEnd Fluorometric TUNEL System kit (Promega, Madison, WI, USA), as recommended by the manufacturer's manual.

Flow cytometry
For flow cytometry, 3 � 10 5 cells were seeded in six-well plates and allowed to attach overnight.On the next day, the complete medium was exchanged for 3PO-containing medium in the desired concentrations.After 24 hours of treatment, cells were harvested using trypsin (Gibco; approx.5-10 minutes at 37 °C), washed with 1� PBS, and resuspended in complete medium containing 50 µg RNAse (Sigma).After 10 minutes of incubation on ice, propidium iodate (PI) was added to a final concentration of 50 µg/mL.Cells were kept on ice and immediately analyzed with LSR Fortessa X-20 Cell Analyzer (Becton Dickinson, USA) using a yellow-green laser with 610/20 nm (ex/em).Interpretation of the data was done using Flow Jo 10.8.1 (Becton Dickinson, RRID:SCR_008520).Singlets were gated via size exclusion, and thresholds were designed with the use of single-stained untreated samples.Graphics were produced using GraphPad Prism 9 (GraphPad Software Inc.).

Seahorse assay
The patient-derived tumor organoid (PDO) was treated with TrypLE (Invitrogen) for 10 minutes at RT, pipetted up and down 10� with a 5-mL serological pipette adapted to a 200 µL pipette tip, and then passed through a 40 µm mash (Greiner Bio One) to obtain a single-cell suspension.Next, cells were manually counted using a Neubauer-improved chamber (Paul Marienfeld GmbH), and approximately 2,000 cells were seeded in 5 µL of Matrigel (Corning) into 96-well spheroid microplates (Agilent Technologies, Santa Clara, CA, USA).Organoids were allowed to grow for 6 days in organoids' medium before treatment with 10 µmol/L 3PO for 24 hours.Before plate reading, a complete medium was exchanged for 180 µL XF serum-free medium (Agilent) supplemented with 1% sodium pyruvate and glutamine (both from Gibco).Seahorse XFe96 instrument (Agilent, RRID:SCR_019545) was used.All injected reagents (glucose, oligomycin, and 2DG) were from Sigma, and their final concentrations in the medium were 250 mmol/L, 30 µmol/L and 250 mmol/L, respectively.Graphics were produced using GraphPad Prism 9 (GraphPad Software Inc.).

Institutional review board statement
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethical Committee of the University Medical Center, Göttingen (UMG # 25/3/17).A written informed consent has been obtained from all subjects.

3PO affects CRC cell proliferation and viability in a concentration-dependent manner
Previously, the cytotoxic effect of 3PO has already been shown for multiple cancer cell lines (31).However, its impact on colorectal cancer remains unclear.Therefore, to characterize the effect of PFKFB3 blockade on colo-  and C).Corroborating the previous findings, LDH release increased after 24 and 48 hours of treatment with 3PO, indicating cell death is enhanced upon inhibition of glycolysis (Figs.1D and E).CRC cells showed to be more susceptible to 3PO at lower concentrations (10 and 25 µmol/L) than normal HUVECs and RPE cells.Among CRC cell lines, both RC cell lines SW-1463 and SW-837 exhibited significantly lower cell viability and increased LDHrelease compared with the HCT-116 and HT-29 cells.It is noteworthy that immunoblot analysis of the colorectal cancer cell lines' lysates did not show a direct correlation between PFKFB3 expression and its inhibition's response by 3PO (Supplementary Fig. S1B).

3PO reduces colorectal cancer cell invasion and endothelial sprouting capabilities
As previously shown, blockade of glycolysis in cancer cells can be associated with cytoskeleton rearrangements, which might impair tumor cell migration as well as tumor cell invasion (18,34).Accordingly, using a Boyden chamber assay, we evaluated the effects of 3PO on cancer cell invasiveness in HCT-116, HT-29, SW-1463, and SW-837 cells.Here, we found a significant decrease in CRC cell invasiveness using a low concentration of 3PO (10 µmol/ L; Fig. 2A).Next, we evaluated 3PO effects on migration by performing a wound healing assay.Time-lapse micrographs and image software analyses were used to monitor the migration of HCT-116, HT-29, and SW-837 cancer cells (Fig. 2B; Supplementary Fig. S1C-S1F).Due to its piled-up growth pattern, SW-1463 cancer cells could not be used for this experimental setup.
Though CRC cell lines SW-837 and HT-29 were significantly affected by 25 µmol/L 3PO (Fig. 2C; Supplementary Fig. S1C-S1D), HCT-116 cells showed no changes in migration capabilities (Supplementary Fig. S1E and   S1F).To further evaluate the molecular changes in cytoskeleton-related genes, we analyzed the mRNA expression of cell invasion-and migrationrelated genes.Corroborating our findings, SW-837 and HT-29 cells treated with 3PO had reduced expression of PAXILLIN, cell division control protein 42 (CDC42), and Wiskott-Aldrich syndrome protein 1 (WASF-1), whereas HCT-116 cells did not show relevant changes of the expression of these genes (Fig. 2D; Supplementary Fig. S1G and S1H).
Notoriously, glycolysis controlled by PFKFB3 regulates migration of endothelial cells.In particular, TECs are characterized by an activated glycolytic phenotype and increased expression of PFKFB3 (14,35).Subsequently, in addition to the motility and invasion effects of 3PO on cancer cells, we also assessed its effects on sprouting and vessel-like formation using endothelial spheroids.Spheroids treated with 3PO showed a significantly shortened sprout length and reduced vessel-like numbers (Figs.2E and F), in association with reduced proliferation by Ki67 IHC staining (Fig. 2G; Supplementary Fig. S1I).Lastly, to reassess the findings by Kim and colleagues (36) that 3PO only prevents sprout initiation but does not cause the regression of established vascular networks, in our experimental setup, we have performed 3PO treatment on established endothelial sprouts for 24 hours and quantified a significant reduction in sprouting length with 20 µmol/L 3PO (Fig. 2H), corroborating our previous findings in vivo (15) and again highlighting model-dependent results.

Patient-derived cancer organoids treated with 3PO show a gene set enrichment of genes related to oxidative phosphorylation
To evaluate 3PO-induced changes on cellular transcriptomes in a more advanced in vitro model, PDOs of three different patients were treated with 30 µmol/L 3PO for 24 hours, and their RNA was extracted and sequenced.As observed in Figs.3A and B, at the end of this time point, 3PO treatment did not affect tumor organoids' morphology or growth.Next, the resulting gene expression data were interpreted by GSEA.Supporting the idea that 3PO treatment would influence a metabolic shift away from glycolysis, among the Gene Ontology collection "Biological Process" (27), 9 out of the 10 most enriched gene sets were associated with oxidative phosphorylation and mitochondrial translation (Fig. 3C).In order to evaluate whether these transcriptomic changes were also detectable at the protein level, we further treated three additional different organoids with 30 µmol/L 3PO for 24 hours and collected protein lysates.Immunoblot analyses for the expression of NDUFB6, a gene found enriched in several of the datasets evaluated (Supplementary Fig.

S2A-S2C
), and ACAD9, a critical influencer on oxidative phosphorylation (37) also found enriched in the GSEA, were also found increased upon 3PO treatment in two of the three PDOs and in three of the four tested colorectal cancer cells (Figs. 3D and E; Supplementary Fig. S3A).Additionally, we were able to see a downregulation of the glycolytic marker LDHA in 3PO-treated PDOs.
Last, we treated for 24 hours PDOs with a lower concentration of 10 µmol/L 3PO (to prevent unwanted cytotoxic effects) and performed a seahorse analysis.
In line with the previous results, we observed that both the basal glycolysis levels and the glycolysis upon glucose stimulation were reduced in two of the three PDOs.Also, a shift in oxygen consumption ratio was observed in two PDOs treated with 3PO (Fig. 3F; Supplementary Fig. S3B and S3C).
Taken together, these findings suggest that 3PO is able to alter the transcriptomic metabolic status of PDOs toward oxidative phosphorylation and reduced glycolysis.

3PO potentializes irradiation effects on colorectal cancer cells
Increased glycolysis is crucial for proliferation and survival of tumor cells (38).As a regulator of glycolysis, PFKFB3 plays a substantial role in this metabolic scenario, and a variety of cancer entities have been characterized by enhanced PFKFB3 expression, including CRC.We have previously shown an association of high expression of PFKFB3 with poor survival of patients with CRC (18).Concomitantly, increased glycolysis is assumed to cause resistance to RT and chemotherapy in several cancer types (39).Therefore, we next evaluated the impact of 3PO and radiotherapy combination in vitro by a colony formation assay.In all tested colorectal cancer cell lines, a significant decrease in cell survival fraction was observed upon exposure to 3PO (Fig. 4A).The administration of 3PO resulted in a significant reduction of clonogenic survival (which assesses cell death upon treatment with ionizing radiation and the potency of cytotoxic agents) in HCT-116 cells, HT-29 cells, and SW-1463 cells as well as SW-837 cells.To further complement our results, CRC cell lines were subjected to a combination therapy consisting of increasing concentrations of 3PO and a single dose of RT (6 Gy).Corroborating our findings, all CRC cell lines showed a significant decrease in the cell survival fraction when 3PO was added to RT in comparison to 3PO alone (Fig. 4B).In order to provide a more detailed assessment, we calculated the CDI (Supplementary Fig. S4A).According to our results the CDI yielded synergistic interactions across all CRC cell lines (CDI <1).In particular, the lowest CDI values throughout all concentrations were exhibited in RC cell lines SW-1463 and SW-837.Irradiation is well known to alter the biological behavior of tumor cells, and several studies have previously indicated that RT can induce tumor invasion and migration, promoting metastasis (40)(41)(42).
Thus, we evaluated the combinatory effects of 3PO and RT on cell invasion and migration of CRC cell lines.As shown in Fig. 4C, RT alone had no influence on cancer cell invasion.In line with our previous results (Fig. 2A), and SW-837 did not show any significant additional decrease in invasiveness in combination therapy when compared with 3PO alone.Nevertheless, when combined treatment was compared with RT alone, a significant decrease in cell invasion was observed across all CRC cell lines, indicating a favorable impact of 3PO in combination with RT.Next, we performed a wound healing assay combining 3PO and RT.RT alone did not alter the migration behavior of HCT-116, HT-29, and SW-837 cells (Figs. 4D-F; Supplementary Fig. S4B-S4D).Indeed, when RT was combined with 3PO, all CRC cell lines showed a significant suppressed migratory ability (Figs.4D-F).Furthermore, assessment of HUVEC viability, sprouting, and vessel formation suggested that combining low doses of 3PO to the RT regimen might also affect tumor vascularization (Supplementary Fig. S4E-S4G).In sum, these findings indicate that 3PO is able to enhance radiation effects.The combination treatment produces synergistic effects in CRC cell lines, resulting in decreased cell survival (in all CRC tested cell lines), cell invasion (HCT-116 and HT-29), and cell migration (HCT-116, HT-29, and SW-837), even at a low concentration of 10 µmol/L 3PO analysis.

3PO administration in combination with radiotherapy increases tumor necrosis in vivo in a rectal patient-derived xenograft mouse model
To confirm our results in vivo, we established an RC patient-derived xenograft (PDX) model.For this purpose, fresh isolated human rectal tumor samples (RC of the upper third; G2; pT4b pN2b cM0) were transplanted into immunodeficient mice (43) for expansion.Subsequently, upon PDX growth, we followed a treatment protocol that simulated clinical conditions, i.e., fractionated radiation doses (1.8 Gy/dose), in combination with i.p.
administration of 25 mg/kg 3PO, which has been shown to be well tolerated at this dose and to induce TVN in the B16-F10 mouse model (14).Mouse cohorts that received RT or the combination of RT and 3PO showed significant weight loss during the treatment (Fig. 5A) but no other signs of systemic toxicity.Tumor growth was not suppressed upon treatment with sham (control group) or 3PO alone, and these cohorts had to be sacrificed due to their increased PDX sizes at days 54 and 62, respectively.Both RT alone and 3PO combined with RT cohorts had their tumor growth affected by treatment.However, 3PO and RT in combination did not further reduce tumor growth when compared with RT alone (Fig. 5B).
Tumor regrowth after radiochemotherapy and clinical complete response is an emerging clinical problem (44).Therefore, we sacrificed the two remaining mice cohorts approximately 30 days after the last RT.During this period, none of the cohorts showed sustained regrowth, evidenced by their tumor sizes and tumor weights (Figs.5B and C).Next, we evaluated histologically all tumor samples, observing that tumors treated with the combination of 3PO and RT had extensive necrotic areas and a difference in tumor viability, when compared with RT-alone tumors (Figs.5D and E).Although cleaved caspase-3 levels were similar in RT and 3PO in combination with RT cohorts, TUNELpositive cancer cells were also significantly increased in the combinatory group (Figs.5F-H).Additionally, we could observe a significant reduction in cell proliferation by BrdU Incorporation, when 3PO was combined with RT (Fig. 5I).Furthermore, PFKFB3 expression levels were found decreased in tumor tissues when 3PO was used alone or in combination with RT (Figs. 5J   and K).
Taken together, these results reveal that 3PO administration lowers PFKFB3 expression in tumor tissue, thereby enhancing RT effects, increasing cell death and reducing RC proliferation in vivo.

3PO administration induces TVN and alleviates hypoxia in vivo
Hypoxia is a prominent obstacle to the efficacy of radiotherapy, and tumor tissue oxygenation plays a crucial role for the efficacy of cancer treatments (45,46).Moreover, no in vivo studies have been performed in human RC PDXs evaluating the use of 3PO for TVN induction, in combination with radiotherapy.
Therefore, to analyze the effects of 3PO administration in combination with RT on tumor vascularization, we have performed a CD31 and αSMA co-staining with our PDX tumor samples (animal cohort from Figs. 5 and 6A).Compared with sham, tumors treated with RT alone showed a significantly reduced CD31 + -αSMA + co-stained area (vessel density), which was partially restored by concomitant treatment with 3PO, leading to comparable vessel density (Figs.6A and   B).Moreover, vessel lumen size increased when 3PO was subjected alone or added to therapy (Fig. 6C; Supplementary Fig. S5A).Further analysis showed that the vessel number decreased significantly for RT and combination therapy of 3PO and RT (Fig. 6D; Supplementary Fig. S5A).Next, tumor hypoxia was assessed by pimonidazole staining, as an indicator of tissue oxygenation [Fig.6E; Supplementary Fig. S5B; refs.(14,15)].Hypoxic areas were reduced when tumors were subjected to 3PO alone or added to RT (Fig. 6F).These results reveal the TVN effects of 3PO in combination with RT in human RC.To further assess the effects of increased tumor oxygenation on the treatment of human RC with radiotherapy, we performed immunohistochemistry staining for γ-H2AX, a marker of DNA double-stranded breaks (Fig. 6G).Although animals were sacrificed 30 days after their last RT treatment, 3PO in combination with RT showed an enhanced γ-H2AX index, indicating the greatest DNA damage out of all treatment cohorts (Fig. 6H).Summarizing, we could observe the therapeutic efficacy of 3PO, through TVN in RT of human RC.

Discussion
In order to adapt to an increased energy consumption, tumors must alter their metabolic phenotype (47).Frequently, cancer cells enhance their glycolytic activity even in the presence of oxygen, an effect described as the "Warburg effect" (48).To maintain their high glycolytic flux, multiple mechanisms might be used by cancer cells to increase their glycolytic flux, which consequently can enhance the expression of genes associated with cell motility, survival, and therapy resistance (18,(49)(50)(51)(52)(53).One, in particular, is the overexpression of the bifunctional enzyme PFKFB3, which plays a crucial role in glucose metabolism (54).PFKFB3 catalyzes the synthesis of F-2,6-BP, leading to intracellular accumulation of F-2,6-BP.As an allosteric activator, F-2,6-BP stimulates phosphofructokinase 1, the most potent stimulator of glycolysis, thereby meeting the requirements for increased energy metabolism of tumor cells.PFKFB3 is overexpressed in several human cancers and promotes proliferation and migration as well as suppresses apoptosis of cancer cells (55).Furthermore, PFKFB3 is known to regulate angiogenesis (35).The endothelium of tumor vessels is characterized by a highly heterogeneous and functionally abnormal structure leading to tumor hypoxia and thereby promoting tumor progression and its dissemination (11).Interestingly, TECs are characterized by an activated glycolytic phenotype and increased PFKFB3 expression.Therefore, interfering the upregulated glucose metabolism in tumor cells and TEC seems to be a promising approach to   hold tumor progression and trigger TVN (14,15).Moreover, this event seems to be possible through PFKFB3 inhibition by 3PO administration.
In this study, we have explored the effects of PFKFB3 inhibition by 3PO in vitro and in vivo using endothelial cells, colorectal cancer cells, PDOs, and PDXs.First, our results are consistent with previous studies showing that 3PO affects cell proliferation and viability of transformed cell lines in a time-and dose-dependent manner (31).Interesting, we observed that among the CRC cells tested, SW-1463 and SW-837 cells showed significantly decreased cell viability and increased LDH release as compared with the other CRC cells (Figs. 1B-E).As PFKFB3 protein expression levels do not always show a direct correlation to 3PO response (Supplementary Fig. S1B, immunoblot with CRC cell lysates), additional reasons for this phenotype upon 3PO treatment might be related with the intrinsic metabolic needs, directly dictated by the mutational status (TP53 mutation; KRAS mutation; CpG island methylated phenotype) and by its transit-amplifying undifferentiated phenotype, which could also influence its susceptibility to 3PO (32,33).Importantly, we cannot exclude the intrinsic differences between cell lines in the expression of PFKFB3 and intracellular F2,6BP levels, which cannot always be directly correlated with glycolytic flux (56).Regarding the selectivity of 3PO cytotoxicity on normal, nontransformed cells, we observed that HUVECs and RPE cells well tolerated the lower in vitro doses, compared with most of the cancer cells.Therefore, we hypothesized that the used normal, nontransformed cells were able to shift more easily from glycolysis to oxidative phosphorylation (OXPHOs) than transformed cells, preventing them from suffering massive cell death.
Next, we showed that treatment of different CRC cell lines with a low dose of 3PO caused a reduction in tumor cell migration and invasion, indicating that blocking glycolysis in cancer cells is associated with cytoskeletal rearrangements (18).Simultaneously, we observed reduced expression of PAXILLIN, CDC42, and WASF-1.Moreover, consistent with the fact that HCT-116 exhibited no reduction in migration by PFKFB3 inhibition, there were no alterations in the expression of those genes in this cell line.Differences in its mutational status might be responsible for the observed results, as HCT-116 is highly hypermutated and microsatellite instable, as opposed to HT-29, SW-837, and SW-1463, which are nonhypermutated and microsatellite stable (32,33,57).This hypothesis is supported by recent proteomic data showing glycolytic differences between microsatellite instable and stable CRCs, which may have not only paracrine but also autocrine effects (58).It is noteworthy that from all four cell lines used in the invasion assay, SW-1463 cells were the most susceptible to 3PO effects on viability, and therefore we cannot exclude that its reduction in invasion is also related to 3PO cytotoxic effects.Furthermore, we have observed that endothelial spheroids subjected to low doses of 3PO show reduced sprouting and vessel-like formation and that this effect could be due to reduced endothelial proliferation.Nevertheless, the effects on the tip cell phenotype cannot be excluded, as it has been suggested that PFKFB3 is also involved in this process (59).
Corroborating the idea that 3PO treatment would efficiently disturb glycolysis in cancer cells, and that CRT resistance could be overcome upon inhibition of glycolysis, we observed a shift in the transcriptomic metabolic flux of patientderived tumor organoids toward oxidative phosphorylation, which denotes a possible compensatory mechanism to account for energy deprivation caused by reduced glycolysis (60,61).Additionally, a subunit of complex I, the largest complex in mitochondrial respiratory chain, NDUFB6 expression is enhanced after treatment with 3PO, and as previously shown by Shi and colleagues (62), CRC cells deficient in complex I exhibited enhanced radiotherapy resistance, along with increased glycolysis.Moreover, the gene set "GOBP Extracellular Matrix Assembly" was found most enriched in the control group, reinforcing previous findings that glycolysis can affect extracellular composition and therefore further influence cell proliferation, adhesion, and migration (18,34,63).Remarkably, by reviewing the most relevant gene sets of 3PO-induced genes on CRC in the protein atlas (NDUFB6, NDUFAB1, NDUFA4, CDC7, CDKN3, PLK4; ref. 64), we found that the increased expression of these proteins correlated with a better prognosis (Supplementary Fig. S6).
Next, we hypothesized that RT resistance could be overcome upon inhibition of glycolysis.Other studies have shown that CRC cells were sensitive to glucose deprivation through PFKFB3 inhibition (18,65).However, in these studies, a radiosensitizing effect through PFKFB3 inhibition on CRC cells remained unclear.Our results demonstrate that PFKFB3 inhibition by 3PO increases radiation-induced cell death of CRC cells while reducing cancer cell migration and cancer cell invasion during RT in vitro.Previously, it has been suggested that 3PO could induce changes in cell cycle (31) and that cancer cells arrested at the G2M phase would exhibit increased sensitivity to irradiation (66).Interestingly, at the used lower 3PO concentrations, we did not observe changes in cell cycle upon 3PO treatment (Supplementary Fig. S7A-S7C), which led us to speculate that these effects were associated (i) with direct changes in the expression of migration/invasion-related genes and (ii) with reduced ability to buffer increased radiation-induced radicals (ROS) upon a shift to metabolic flux toward oxidative phosphorylation, possibly associated with reduced accumulation of glycolytic products, such as glutathione and pyruvate (67).
Considering our in vitro results, we decided to evaluate the treatment response of human RC PDXs to 3PO in vivo, as it has been already shown that 3PO administration at lower doses does not cause severe systemic effects in mice (15,31,68).Previously, we showed that in a mouse model of murine melanoma, 3PO treatment at low concentrations triggered TVN (14).Now, using human patient-derived RC xenografts, we demonstrate that tumors treated with concomitant administration of 3PO and RT exhibited reduced hypoxia, suggesting that also in RC, the inhibition of PFKFB3 by 3PO leads to a similar outcome.This finding was supported by enlarged vessel lumina through CD31 staining upon 3PO treatment and changes in tumor vessel density.Interestingly, this result also suggests that in mice, at least partially, TVN can be achieved without T cells' mediation, a finding previously discussed by Lian Tian and colleagues (69).Of clinical relevance, further evaluation of 3PO effects at low doses on immune cell function will be necessary, as many linages like T cells require glycolysis for their proper activation (70).
Regarding tumor response to RT, we show that administration of low doses of 3PO in combination with RT resulted in extensive tumor necrotic areas when compared with control groups.This effect was associated with increased tumor DNA damage and cell death, evidenced by both increased H2AX phosphorylation and TUNEL-positive tumor cells.These results are in line with the report that PFKFB3 inhibition can directly affect DNA damage repair and homologous recombination upon radiation (71).Moreover, increased DNA damage can also be attributed to an increase in tumor oxygenation levels due to TVN, which further triggers ROS-mediated tumor cell death.Importantly, we have not observed macroscopic differences in tumor sizes between single RT and combined 3PO + RT cohorts (Fig. 5B).
This result might be related to pronounced tumor sensitivity to RT and/or the dose of administrated RT.Thus, further studies using different doses of
Fig.S1A).Additionally, to directly assess cell viability and cell death upon 3PO treatment, we performed CellTiter-Blue assays and LDH-release assays with CRC cell lines as well as with normal, nontransformed cells.A clear time-and dose-dependent reduction of cell viability was observed after 24 hours of treatment with 3PO, and it was intensified after 48 hours (Figs.1B and C).Corroborating the previous findings, LDH release increased after

FIGURE 2 FIGURE 3 FIGURE 4
FIGURE 2 3PO reduces colorectal cancer cell invasion and endothelial cell sprouting capabilities.A, Invasion assay performed with HCT-116, HT-29, SW-1463, and SW-837 cancer cells.Cells were plated in a Boyden multi-well chamber and treated with 10 µmol/L 3PO for a period of 96 hours.Data are displayed as means SD, n ¼ 3. * * , P < 0.01; * * * * , P < 0.0001; Student t test.B, Representative pictures of migration assay performed with SW-837 treated with 10 and 25 µmol/L 3PO; 0 and 30 hours after removing culture inserts.C, Cell migration assay quantification of SW-837 treated with different concentrations of 3PO for 48 hours.Data are displayed as means ± SD, n ¼ 3. D, Relative mRNA expression levels of PAXILLIN, CDC42, and WASF-1 in SW-837 cells treated with different concentrations of 3PO after 72 hours.Data are displayed as means SD, * , P < 0.05; * * , P < 0.01; oneway ANOVA.E, Representative pictures of cell sprouting assay performed with HUVECs treated with 10 and 20 µmol/L 3PO after 0 and 24 hours.F and G, Sprouting assay quantification of HUVECs upon PFKFB3 inhibition.Sprouting length, vessel number, and EC proliferation analyzed upon treatment with 10 and 20 µmol/L 3PO for a period of 24 hours.H, Sprouting length quantification upon 24 hours of 3PO treatment on already established spheroids.Data are displayed as means SD, n ¼ 3. * * * , P < 0,001; * * * * , P < 00,001; one-way ANOVA.