In this study, we report a differential response of mitogen-activated protein kinase–kinase (MEK) inhibitor trametinib in 20 head and neck squamous cell carcinoma (HNSCC) patients’ tumor-derived cell cultures. Relatively sensitive and resistant cases to trametinib were identified using high throughput metabolic assays and validated in extended dose response studies in vitro. High throughput metabolic assays exploring combination therapies with trametinib were subjected to synergy models and maximal synergistic dose analyses. These yielded several candidates, including axtinib, GDC-0032, GSK-690693, and SGX-523. The combination regimen of trametinib and AXL/MET/VEGFR inhibitor glesatinib showed initial efficacy both in vitro and in vivo (92% reduction in tumor volume). Sensitivity was validated in vivo in a patient-derived xenograft (PDX) model in which trametinib as a single agent effected reduction in tumor volume up to 72%. Reverse Phase Protein Arrays (RPPA) demonstrated differentially expressed proteins and phosphoproteins upon trametinib treatment. Furthermore, resistant cell lines showed a compensatory mechanism via increases in MAPK and non-MAPK pathway proteins that may represent targets for future combination regimens. Intrinsic-targeted options have potential to address paucity of medical treatment options for HNSCC cancer patients, enhance response to extrinsic targeted agents, and/or reduce morbidity as neoadjuvant to surgical treatments. The 8-year survival of HNSCC is approximately 30.2% for HPV negative disease and approximately 70.9% for HPV positive disease.5 In this study, we focus on HPV negative disease, which is more aggressive. The standard treatment for HNSCC largely depends on tumor location, TNM staging, and medical comorbidities. Treatment incorporates surgery, chemotherapy, and radiotherapy singly and in combination. Chemoradiotherapy is the standard of care in unresectable, locally advanced disease.4 Thus far, two PD1 targeted immunotherapies, pembrolizumab and nivolumab, and one tumor intrinsic EGFR targeted therapy, cetuximab, are FDA approved for treatment of HNSCC.6Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide, resulting in 450,000 deaths annually, with 10,030 deaths in the United States.8 Additionally, cetuximab, the only tumor-intrinsic targeted therapy, was approved for use in combination with radiotherapy.9 However, cetuximab has declined in favorability as radiotherapy alone is no longer the standard of care.5 When cetuximab is used in combination with chemotherapy in the recurrent/metastatic setting, it extends progression-free survival (PFS) and overall survival for less than 3 months.9 Unfortunately, cetuximab also carries multiple side effects, resulting in an increase in adverse events.5 These limited therapeutic options represent a paucity in targeted therapeutics available for the treatment of advanced HNSCC, underscoring a need for new approaches.Although the introduction of new immunotherapies has greatly expanded HNSCC treatment options, only 13–18% of recurrent or metastatic cases are responsive, with all but 5% ultimately progressing to death.BRAF V600E or V600K mutation is present. In melanoma, trametinib single agent increased PFS by 4–7 months in BRAF V600E mutant tumors.10 Interestingly, there was a partial response rate of 10% in BRAF wild-type tumors, demonstrating that a subset of non-BRAF mutant tumors may benefit from MAPK inhibition. In HNSCC, Phase II randomized clinical trials with single-agent trametinib have shown modest results, with >25% reduction in tumor volume in 11/17 patients treated prior to tumor resection.11While estimates vary, the RAS-MAPK pathway has been found to be mutated in as high as 18% of HNSCC.6 Our underlying hypothesis is that patient-specific tumor intrinsic targeting has a role in improving therapeutic options and treatment efficacy. In this study, we focus on the more aggressive HPV negative HNSCC, examining trametinib as a therapeutic agent across HNSCC patient-derived primary and early secondary cell cultures; demonstrate patient-specific efficacy in vivo; and identify potential combination therapies that may synergize with trametinib treatment.MEK inhibitors targeting the MAPK pathway are used in combination with BRAF inhibitors in melanoma, metastatic lung cancer, and metastatic anaplastic thyroid cancer when a Primary tumor cell cultures derived from 20 HNSCC patients were subjected to a drug screen of 200+ inhibitors, natural products, and combinations . Trametinib was identified as showing selective sensitivity and resistance in the HNSCC cohort, demonstrating a non-uniform metabolic rate profile across cases . For the12 Additionally, trametinib, but not TAK-773 (a MEK1 inhibitor), showed reductions in metabolic rate, demonstrating that both MEK1 and MEK2 inhibition are necessary for cellular response at clinically relevant concentrations . Notably, sensitive case 10292 carries an activating HRAS mutation. HRAS mutations in non-HNSCC cancer cell lines have previously shown sensitization to MAPK inhibitors.13 In cell line 10336, many inhibitors, including trametinib, resulted in increases in metabolic rate, highlighting the importance of stratifying patient populations by drug sensitivity.To confirm selective sensitivity, relatively sensitive cell lines 10004 and 10292 and resistant/insensitive cell lines 10336, 10326, and 20003 were further characterized using an extended dose range in triplicate experiments, confirming case-specific dose-dependent inhibition . Cell li14 Bliss beta coefficients were calculated using SynScreen high throughput combination software.15 Chou-Talalay method for analyzing combination indices yielded similar results to the Bliss beta calculations, with a − .65 Spearman correlation16 . Furthermore, the Combination index (CI) methodology allowed for the identification of regimen dosing that yielded the minimum CI value across the drug dose ranges.16 This allowed us to identify both the synergistic drug targets and the optimal synergistic drug dosing regimens (Supplementary Table S1).After a preliminary screen of trametinib in binary combinations with 150+ other drugs and natural products, we identified and selected synergistic and antagonist drug combinations to be further validated (data not shown). The combination panel was further expanded using drugs with more specific drug interactions to characterize drug effects and pathways. To analyze the combination synergies, we used two different models for analyzing combination synergies, the Bliss independence model and the Loewe additivity model.The Bliss beta values show a normal distribution around the Bliss additivity value of 1, with sensitive cell lines accounting for the majority of the synergistic combinations . Stratifying by cell line, 10336 is identified as being particularly resistant to multiple inhibitors, skewing the distribution of the resistant cell lines . GDC-0032, a PIK3CA inhibitor, was synergistic in 5/6 cell lines, reduced metabolic rate by up to 91.5%, and sensitized 2/3 of the resistant cell lines to trametinib treatment .Figure Trametinib induced a dose-dependent decrease in the phosphorylation levels of ERK regardless of the sensitivities of the cells . This suggests that while trametinib is effective at blocking MAPK pathway inhibition, only a subset of cells will experience a decrease in metabolism. In the sensitive line, 10004, and the resistant line, 10336, trametinib treatment induced an increase in Phosho-AKT levels . This could partially explain trametinib synergies between PIK3CAi, AKTi, VEGFRi, METi, and AXLi, all of which either directly target or target an upstream regulator of Phospho-AKT.In3 to 82 mm3) (3 to 29 mm3), compared to trametinib alone . Although glesatinib alone showed little effect as a single agent in vitro, suggesting that the combination regimen and not glesatinib as a single agent is responsible for the tumor response, the absence of a glesatinib single-agent control in vivo is limitation of our in vivo study would need to be addressed in follow-up studies .To evaluate the relationship of trametinib sensitivity in vitro to efficacy in vivo, we established a patient-derived xenograft (PDX) model using patient-specific primary tumor cells. Trametinib significantly decreased tumor volume in 10004-PDX models  . While nib alone . Remissiib alone . The inh18 The relevance of the PDX model in our study is based upon several lines of evidence. The epithelial tumor cell morphology was indistinguishable in the patient-derived primary cultures and the mouse tumor derived primary culture of that patient after reestablishment of the PDX tumors in culture. Additionally, vimentin-keratin staining of the cultured cells showed the same epithelial to mesenchymal transition (EMT) characteristics in both the PDX and original tumor: 100% epithelial with 4% mesenchymal characteristics. Similarly, tumor pathology of the original case and the PDX tumor from that case were similar based upon H&E staining of tissue sections from each tumor . Finally, the cells from the mouse and primary tumor exhibited similar drug sensitivities .PDX models have been shown to be reliable in terms of replication of original tumor and predicting drug sensitivities .20 Basal proteomic levels of cell lysate showed no significant correlations with drug response.Reverse Phase Protein Array (RPPA) was conducted in order to determine the molecularly and functionally relevant phosphoproteomic changes in response to MEK inhibition. Lysate collected from cells treated with trametinib or vehicle for 24 hours revealed that maximum ERK inhibition occurred at 100 nM regardless of the functional response to trametinib, consistent with other studies .To test the functional relevance of MEK inhibition, lysate was collected from cells treated with trametinib or vehicle at the IC50 value for 3 days. The resistant cell lines were treated at maximum dose of 312 nM. Prior to RPPA analyses, immunoblot and metabolic rate assays confirmed the expected functional and molecular responses, ensuring the integrity of the cell lines and analyses . Both 24-hour and 3-day treatment samples were collected and sent to MD Anderson for RPPA analyses.All trametinib-treated cell lines showed a significant decrease in MAPK phosphorylation at T202 and Y204 . Although the MAPK phosphorylation antibody used for these results was designated as “use with caution” and therefore not used in the primary analysis, the decreases in MAPK phosphorylation indicates that trametinib was effective in inhibiting its primary target.Only MD Anderson-validated antibodies for differential affinity analyses between 24-hour and 72-hour groups . The 72-22 Together, these proteomic changes, specific to the resistant group, are suggestive of a MAPK reactivation mechanism available to trametinib-resistant cell lines. Phosphorylated heat shock protein was differentially increased in both resistant and sensitive groups, suggesting cellular stress in both resistant and sensitive cell lines. CDC25C, DNMT1, and RB1 phosphorylation were decreased across both resistant and sensitive cell lines at 72-hours. AURKB, CDC6, CCNB1, and FOXM1 showed differential decreases in resistant cell lines at both the 24-hours and 72-hour time points but failed to reach significance in the sensitive cell lines.When comparing between resistant and sensitive cell lines, the resistant groups showed a greater number of protein antigens differentially expressed at both the 24- and 48-hour time points, compared to the sensitive groups . Eight g11 These mixed results likely relate to the complexity and heterogeneity of HNSCC. HNSCC is a particularly diverse cancer in terms of molecular classification, biological characteristics, genomics, and HPV status, as well as in anatomical sites.23 Our exploration into the landscape of combination therapies offers the opportunity of increasing treatment efficacy amongst this heterogenous population of patients.24 Rational optimization of trametinib therapy for HNSCC will be enhanced by further evaluation of the differential targets in HNSCC, pathways underlying sensitivity or resistance, dose selection, and identification of promising synergistic combinations. This approach to new targets and pathway synergies is relevant to other cancers.In this study, we show that within a cohort of patients’ HNSCC derived primary tumor cell cultures, trametinib showed selective sensitivity in reducing tumor metabolic rate in a subset of HNSCC. Recently, trametinib as a treatment for HNSCC has been supported by clinical data as the result of the Phase II randomized clinical trial of trametinib in HNSCC showing mixed results, with observed reduction in tumor volume in 65% of patients.26 We also observe a statistically significant increase in AKT phosphorylation at 72 hours. This is concordant with dose response immunoblotting on the patient-derived cell lines . The increase in AKT phosphorylation is a plausible mechanism behind the synergies observed in the PIK3CA and AKT inhibitors in combination with trametinib.In order to further enhance the response of trametinib, we evaluated the synergistic response of both trametinib-sensitive and trametinib-resistant HNSCC patient-derived cell lines. Many cell lines showed drug synergies between VEGFR, PIK3CA, AXL, AKT, and MET inhibition. Using MAPK synergies by co-targeting the AKT, MTOR, and PIK3CA has shown promise in other HNSCC studies.27 However, despite the observation of synergy between AXL/MET inhibition and trametinib combination treatment, there seemed to be a reduction in the overall AXL and MET expression in trametinib-treated cells, suggesting that the cells that are sensitive to trametinib may be the same cells that highly express AXL and MET . Alternatively, trametinib may lead to the down regulation of AXL and MET in the context of HNSCC.An additional area of interest is the Receptor Tyrosine Kinase (RTK) response in combination with Trametinib, many of which are upstream regulators of the PIK3CA and AKT pathways. AXL and MET have been suggested to increase resistance of MAPKi and have shown synergies with MAPKi in non-HNSCC models.28 By identifying the therapeutic window that yields the maximum synergistic benefit and minimum CI value, we identified optimum dosing regimens across many combination therapies (Table S1). Identification of optimal dosing ranges is critical due to off target effects of many of the inhibitors, as well as for limiting drug toxicity.29Interesting, the cell line demonstrating the most EMT, 10336, was only synergistic with FGFR inhibition.30Additionally, we show that response in vitro correlates to a decrease in tumor volume in vivo. This is especially promising due to the concordance between the primary tumor and the PDX established tumor. Furthermore, our data suggest that treatment durability may be improved by the addition of a synergistic agent, in this case glesatinib. Glesatinib targets MET, TIE-2, AXL, as well as VEGFR 1/2/3. It is unclear which of these signaling pathways may be responsible for a more durable response. Whereas trametinib decreased the concentration of both MET and AXL on the cellular surface in our cell lines shown by immunofluorescence , VEGFR1 showed significant increased affinity at 72 hours in the RPPA data. The implications of this finding remain to be elucidated; however, VEGFR1 as a plausible molecular mechanism behind this synergy and needs to be substantiated in further studies.31These exploratory RPPA studies identified many proteins upregulated in response to trametinib treatment that are targetable, including DDR1, LCK, MMP14, PAI-1, SOD, CD26, and FAK, that could be evaluated for future trametinib combination therapies. It was of interest to observe that trametinib-resistant lines had an increased adaptive proteomic response to trametinib treatment, including the activation of the MAPK pathway though increase of p-MEK1 and GAB2. The totality of these observations is suggestive of an adaptive response to trametinib allowing for resistant cell lines to survive MAPK inhibition. The primary culture conditions selected for epithelial HNSCC tumor cells without fibroblast or immune cells. Both a limitation and an advantage of this approach is that the RPPA data reflect the intrinsic epithelial tumor cells. While this allows us to dissect the intrinsic pathways of the patient’s individual HNSCC, it lacks the context of the tumor microenvironment. Recently, trametinib has been reported to enhance efficacy of PD-L1 inhibitors in HNSCC. Thus, besides combinations for intrinsic targeting, trametinib could be combined with already approved immunotherapies to increase treatment efficacy in HNSCC.While this study yielded several promising candidate drug combinations for trametinib, a major limitation remains biomarkers predicting who will and will not respond to trametinib or trametinib combination therapies. This is especially important considering that trametinib enhanced metabolism in a subset of primary cultures within our HNSCC patient cohort . In our 33 Further preclinical studies on these combinations in more complex immunocompetent models and on predictive biomarkers in larger cohorts is warranted to extend the therapeutic options of HNSCC patients by applying promising regimens in the clinic.A major limitation of the study is inability to address the potential complementary role of intrinsic and extrinsic targeting or how intrinsic pathways may affect the microenvironment or immunotherapy responses, given our focus on the epithelial tumor cell component in culture, and our use of an immunocompromised animal model for in vivo assessment of drug response.34 Primary tumor samples were collected from the operating room in DMEM/F12 Media, supplemented with 2x antibiotic/antimitotic . Tissues were washed 3 times in fresh DMEM/F12 Media and minced before plating. Cells were cultured in DMEM/F12 Media , supplemented with 5% supplemented BCS (Hyclone), 1x antibiotic/antimitotic , 1.8 × 10–4 M adenine , 0.4 μg/mL hydrocortisone (Sigma), 1 × 10–10 M cholera enterotoxin (Sigma), 2 × 10–9 M triiodothyronine (Sigma), 5 μg/mL insulin (Sigma), and 10 μg/mL epidermal growth factor (Invitrogen). Fibroblasts were removed from primary cultures by differential trypsinization using Trypsin-EDTA (0.25%), phenol red . Frozen cultured cell lines were used within five passages of the original tumor.Primary tumor cell lines were obtained from patients treated at the OHSU Department of Otolaryngology for HNSCC from 2014 to 2019 . Cell culture methods have been previously described.At time of selection, sensitive cases that were <20% of the median IC50 values were selected for validation. Trametinib  was plated using the ECHO liquid handler in a 13, three-fold dilution series ranging from 10 μM to .01 nM. Cell lines 10004, 10292, 10336, 10326, and 20003 were randomized by column and plate order and plated in triplicate. Cells were assessed for metabolism after 72 hours of incubation at 37°C using atetrazolium-based metabolic rate assay  by measuring absorption at 490 nM using a Biotek Synergy H1 Microplate Reader. Values were then normalized to wells that received no drug. Dose response curves were created for area under the curve (AUC) analysis.Drug combinations were chosen based on preliminary combination data, drugs with specific targeted pathways, and targets from siRNA data. Trametinib-combination therapies were chosen for further validation on custom 7 × 7 drug combination matrices created using ECHO liquid handler. Trametinib started at 0.03 μM  and followed 6 three-fold dilutions. Cells were assessed for metabolism after 72 hours of incubation at 37°C using atetrazolium-based metabolic rate assay  by measuring absorption at 490 nM using a Biotek Synergy H1 Microplate Reader. Values were then normalized to wells that received no drug.36In parallel to the Bliss beta coefficients calculated by the SynScreen program, we evaluated synergy using the Chou-Talalay Combination Index (CI).Where 37 using a gradient-descent optimizer. Combination dose–response observations were initially modeled as a multidimensional logistic regression; however, after manual inspection, we noticed that the combination-agent dose–response surface exhibited complex behavior that the logistic model was not capable of explaining. To address this, we chose a non-parametric Gaussian process (GP) model.39 Manual inspection of the GP explained more variance in the dose-response without overfitting within the measured dose-range. The GP parameters were chosen using evidence lower bound (ELBO) and a gradient-descent optimizer.Single-agent dose–response curves were fitted with the Hill equationSeveral single-agent inhibitors did not create the desired effect (50% inhibition) within the measured concentration range. To address this, single agents that did not reach 50% inhibition were assigned the inequality:Where If the desired effect (50% inhibition) was not reached in combination screen, then no CI values were calculated.TM or Clarity MaxTM Western ECL Substrate  and imaged using a Bio-Rad ChemiDoc touch MP Imaging System. For all western blots the same blot was probed repeatedly.Patient-derived tumor cells were treated with cell lysis buffer , complete mini protease inhibitor mixture tablets , and PhosSTOP Phosphatase Inhibitor Cocktail Tablets . Lysates were spun at 8,000 xg for 10 minutes at 4°C, mixed 3:1 with 4x Laemmli Sample Buffer  with b-ME, and heated at 95°C for 5 minutes. Lysates were run on Criterion TGX Precast Midi Protein Gel , transferred to a polyvinylidene difluoride membrane , and blocked for 1 hour in TBS-T with 5% milk. Blots were probed overnight at 4°C with PERK (C33E10) Rabbit, Phospho-p44/42 MAPK (Erk1/2) Rabbit, Phospho-Akt (Ser473) (D9E) XP Rabbit, Akt (pan) (C67E7) Rabbit, or a-Tubulin (DM1A) Mouse . HRP conjugated secondary antibodies, anti-rabbit, or anti-mouse IgG were used for rabbit and mouse primary antibodies, respectively. Blots were developed using Clarity2/2.39,40 Mice were randomized using R, into vehicle, trametinib, and trametinib + glesatinib treatment groups. Treatment of 1 mg/kg/day trametinib  (n = 6) or 60 mg/kg/day glesatinib  and 1 mg/kg/day of trametinib (n = 6) or vehicle (DMSO) control (n = 8) was delivered via oral gavage once tumors reached 350 mm3. Glesatinb treatment was lowered from 60 mg/kg/day (weeks 8–10) to 40 mg/kg/day (week 10), to 15 mg/kg/every-two-days (week 11), and finally down to 9 mg/kg/day every-two-days (week 13) after combination toxicity was observed. Three mice died during dose de-escalation in the combination group. All mouse cohort number changes are displayed in 3. Animals were housed within the same treatment group to avoid treatment contamination. Groups were non-blinded throughout the experiment and analyses. Graph pad prism was used to perform student’s t-tests and to generate figures. All studies were performed according to guidelines approved by OHSU Institutional Animal Care and Use Committee.20 female NOD SCID Gamma (NSG) mice (8–16 weeks old) were used for this study . Mice were inoculated with 2 million 10004 HNSCC cells in right flank, and measurements of tumor growth were taken daily. Tumors were measured, and volume was calculated by Length*WidthCells plated in 12-well vessels on collagen coated coverslips, fixed with 4% formalin solution at room temp for 15 minutes, washed twice with PBS, permeabilized in PBS + 0.1% TritonX. Cells were incubated overnight at 4°C with primary unconjugated rabbit cytokeratin 5 , mouse vimentin (abcam-ab8978), and Guinea pig cytokeratin 8/18  in blocking buffer composed of 2% BSA, 5% goat serum, and PBS. Cells were washed three times in PBS-T and incubated with mouse, rabbit, and goat secondary antibodies labeled with 488 GFP, 594 Texas, 647 Cy5, respectively, and diluted 1:300, along with a DAPI. Cells were washed twice with PBS-T and imaged with the 10x objective on the EVOS microscope. Image segmentation, storage, and data analyses were performed using Image J software.41 Cells were grown in 6-well dishes, and cell lysate was collected when cells reached confluence of 50–90%. Lysate buffer and SDS were purchased from MD Anderson. Cell lysate was immediately diluted to ~1 μg/μl and stored at −20°C prior to shipment to MD Anderson. Immune blots of excess lysate confirmed molecular response. Cells plated in triplicate in 96-well dish confirmed functional response after incubation for 72 hours using Promega MTS assay.Cell lysate was collected using RPPA lysis buffer provided by MD Anderson Cancer Center RPPA Core and following their collection protocols.https://www.mdanderson.org/research/research-resources/core-facilities/functional-proteomics-rppa-core/antibody-information-and-protocols.html. For all analyses, we considered the “validated” antibodies only, as defined by MD Anderson and used the normalized log2 values.41 We performed a differential affinity analysis comparing trametinib versus DMSO for both the 24-hour and 72-hour treatment groups. We used the Bioconductor package limma and defined an antibody to be significant if the FDR < .05 using the Benjamani-Hochberg correction.42 In addition, we performed differential affinity analysis comparing samples treated with DMSO and trametinib separately for resistant and sensitive patients. We used the Bioconductor package limma and defined an antibody to be significant if the FDR < .05 using the Benjamani-Hochberg correction.42The MD Anderson RPPA Core Facility performed the RPPA and processed all data. Full annotation of the antibodies can be found at the MD Anderson RPP Core Facility website: Oral and written patient consent was obtained under OHSU IRB protocol 10071, and work was performed under OHSU IRB protocol 809. All animal experimentation was approved by the Institutional Animal Care and Use Committee (IACUC) of OHSU protocol 1489.Click here for additional data file.


We further review recent advances in the understanding of GVHD as a complex immune phenomenon between donor and host immune systems, particularly in the early stages with mixed chimerism, and potential novel therapeutic approaches to prevent the development of GVHD.Immune checkpoint inhibitor therapies and allogeneic hematopoietic cell transplant  represent two distinct modalities that offer a chance for long-term cure in a diverse array of malignancies and have experienced many breakthroughs in recent years. Herein, we review the CD27-CD70 co-stimulatory pathway and its therapeutic potential in 1) combination with checkpoint inhibitor and other immune therapies and 2) its potential ability to serve as a novel approach in graft- However, despite the obvious life-saving benefits of alloHCT, graft- disease , 2. The  disease . These iThe framework of classical acute GVHD occurring in the first 100 days of transplant due to alloreactivity driven by donor T-cells has more recently been supplanted by a more robust understanding involving the intricate interplay of donor and host immune cells with host tissue . The iniAnother realm of treatment modality in the arena of cancer therapy that has revolutionized the field has been the adoption of CPI therapies, which are now utilized in the treatment of a diverse array of advanced stage malignancies, from non-small cell lung cancer to classical Hodgkin’s lymphoma , 20. DesWhile CPI targeting agents derive their function by countering an inhibitory signal, an alternate and possibly synergistic approach has been agonizing T-cell stimulatory co-signaling pathways. Co-stimulatory pathways are broadly speaking, either part of the B7/CD28 or tumor necrosis factor (TNF) family . Clinicaversus-tumor (GVT) effect as there is a strong correlation between incidence and severity of GVHD and disease free survival  have been utilized in the development of both experimental and commercially available second generation chimeric antigen receptor T-cell (CAR-T) therapies leading to significantly greater activation, expansion, and persistence of CAR-T cells , 31. Morsurvival .Thus, it is critical to identify co-stimulatory pathways which when blocked decrease GVHD but do not interfere with GVT. One potential way to decrease the incidence of GVHD would be by inhibiting a co-stimulatory receptor thereby attenuating CD4+ and CD8+ cytotoxic T-cell activity. CD26 has been studied in both pre-clinical and clinical models, while the CD27-CD70 pathway has been studied extensively in pre-clinical murine models. In murine models, inhibition of CD26 [also known as dipeptidyl peptidase-4 (DPP4)] by a monoclonal antibody has been demonstrated to decrease GVHD incidence without compromising GVT . In a smHerein, we conduct an in-depth review of the CD27-CD70 pathway and its application in both GVHD attenuation following alloHCT and its use in the treatment of numerous malignancies in combination with CPI therapies.via Traf2 and Traf5 signaling and activation of the NF-κB pathway  and progenitor cells –40. CD27 pathway . The int pathway . However pathway . Althoug pathway , 49.Under pro-inflammatory conditions  CD27-CD70 activity is increased, leading to proliferation and survival of lymphocytes with multiple downstream effects . CD27-CDin vitro models, CD27 agonism of bone marrow progenitor cells decreased monocytic differentiation and overall inhibited leukocyte differentiation, while in competitive transplantation assays CD27 agonism decreased donor B and T lymphocytes, suggesting the CD27-CD70 pathway’s ability to influence hematopoiesis and immune cell differentiation , glioblastoma, melanoma, renal cell carcinoma, prostate adenocarcinoma, colorectal adenocarcinoma, and ovarian cancer  treated with varlilumab revealed that both CD4+ and CD8+ T-cells were stimulated , which was accompanied by upregulation of other co-stimulatory pathways  along with the inhibitory PD1 pathway , has resulted in varying degrees of objective clinical responses in a subset of cancer patients enrolled. This has included complete remission in Hodgkin’s lymphoma and partial responses in ovarian, colorectal, and squamous cell cancer of the head and neck see Table 1. pathway .CD27 agonism alone and with an PD1 checkpoint inhibitor has also been explored as a potential mechanism of increasing the efficacy of tumor-specific peptide vaccines by enhancing CD4+ helper T-cell and CD8+ cytotoxic T-cell response following vaccination , 74. CliWhile varlilumab has yet to obtain an FDA indicated approval for use, six clinical trials with varlilumab are actively recruiting patients with B and T-cell lymphomas, neurologic malignancies, melanoma, and non-small cell lung cancer Table 1.Traditionally, the prevailing thought behind the etiology of GVHD rested solely with donor immune cells, particularly T-cells becoming activated upon alloreactivity to host antigens. However, more recently, the complex interaction between donor and host immune systems leading to GVHD has been noted, particularly in the early stages of alloHCT, where a mixed chimerism exists , 76. WhiThe most commonly employed conditioning regimens in alloHCT are given with myeloablative or reduced intensity/non-myeloablative intensity consisting of a combination of myelotoxic chemotherapeutic agents with or without TBI –80. The Collectively, these findings suggest the importance of alterations in the bone marrow microenvironment following the noxious insult of the conditioning regimen leading to emergency hematopoiesis and the complex interaction of host and donor immune cells which may persist for many months following alloHCT, during the time acute GVHD is most likely to occur.via caspase-dependent T-cell apoptosis and upregulation of inhibitory immune checkpoint inhibitor pathways , R01HL135325 (XC).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Bone metastases are typically associated with a short-term prognosis. Photodynamic therapy (PDT) emerges as a promising alternative treatment for targeting metastatic lesions. In this study we investigated the effect of 5-aminolevulinic acid-mediated PDT treatment on both primary and human bone metastatic cancer cell lines. We found that human cell lines have different sensitivity to the same doses and exposure of 5-ALA PDT resulting in two different cell fates, apoptosis or senescence, depending on the extent of the cellular damage. As such, PDT has potential applicability in bone metastases of invasive ductal carcinoma.Bone is a frequent site of metastases, being typically associated with a short-term prognosis in affected patients. Photodynamic therapy (PDT) emerges as a promising alternative treatment for controlling malignant disease that can directly target interstitial metastatic lesions. The aim of this study was to assess the effect induced by PDT treatment on both primary (giant cell bone tumor) and human bone metastatic cancer cell lines . After 24 h post light delivery (blue light-wavelength 436 nm) with 5-aminolevulinic acid, the effect on cellular migration, viability, apoptosis, and senescence were assessed. Our results showed that bone metastasis derived from breast cancer reacted with an inhibition of cell migration coupled with reduced viability and signs of apoptosis such as nuclei fragmentation following PDT exposure. A limited effect in terms of cellular viability inhibition was observed for the cells of giant cell bone tumors. In contrast, bone metastasis derived from renal carcinoma followed a different fate—cells were characterized by senescent features, without a notable effect on cell migration or viability. Collectively, our study illustrates that PDT could act as a successful therapy concept for local tumor control in some entities of bone metastases. The incidence of bone metastases in patients with advanced tumor disease has increased in recent decades. After the lung and liver, bone is the third most affected organ by tumor metastases . The cliPhotodynamic therapy (PDT) has been emerging as a promising way to treat and control malignant diseases ,6,7. TheDespite the fact that PDT is used in brain ,17, lungIn this study, we aimed to investigate the effects of PDT treatment on both primary and human metastatic cancer cell lines. Particularly, we investigated both qualitatively and quantitatively the effect of PDT exposure with 5-ALA on cellular migration/invasiveness potential, viability, apoptosis, and senescence growth arrest.Two primary bone metastases cell lines and one primary bone tumor cell line were subjected to investigations. Cell lines originating from primary bone metastases were as follows: ”MAM”—a cell line derived from bone metastases of renal cell carcinoma, and ”MAC”- bone metastases of invasive ductal breast carcinoma. Cell lines originating from a primary bone tumor, “17-1012”—giant cell tumor of bone, were also employed. All cell lines were kindly provided by Dr. Sabine Schleicher from the University Children’s Hospital Tuebingen. The patients  had given their written informed consent to the scientific analysis and cell line establishment from tissue samples. Full Institutional and ethical approval was obtained before the commencement of the study (project no. 008/2014BO2). As controls, bone marrow-derived mesenchymal stem cells (MSC) were isolated as previously described  at the Uv/v) fetal bovine serum  and 2 mM L-glutamine  and MAC and MSC cells in Dulbecco’s modified Eagle’s medium (DMEM) with GlutaMAX, 4.5 g/L D-glucose  supplemented with 10% (v/v) FBS . Cells were cultured at 37 °C in a humidified atmosphere with 5% CO2.Cell lines: MAM, 17-1012 were cultured in RPMI  supplemented with 10% .4 cells—MAC, 1 × 104 cells—MAM, 3 × 104—17-1012, and 2.5 × 104—MSCs cells was seeded into each 2-chamber culture inserts of 6-well plates with opaque F-bottom . After 24 h incubation at 37 °C, 5% CO2, the insets were removed, and the culture medium was replaced with a new one containing either 1 mM or 1.5 mM of 5-aminolaevulinic acid (5-ALA) photosensitizer. The cells were subjected to PDT exposure—blue light  in a continuous output mode for predefined time frames of 300 s and 2000 s. Cells that were not subjected to PDT exposure served as controls.An amount of 2 × 10w/v) paraformaldehyde in phosphate-buffered saline  for 30 min, and stained with 0.5% (w/v) Coomassie Brilliant Blue R250  in 100% (v/v) methanol  for 30 min. The migration of cells into gaps was visualized and photographed using an inverse phase-contrast microscope  and quantified using the open-source software ImageJ . The experiments were repeated three time for each cell type and exposure condition.Following PDT exposure, the cells (see section: “PDT exposure with 5-aminolaevulinic acid”) were fixed with 4% . Principally, cell viability determination was based on the mitochondrial conversion of MTS to soluble formazan, indicative of the number of viable cells [4 cells for each cell type) were supplemented with 50 μL of a 2 mg/mL solution of MTS in complete media and incubated for 90 min at 37 °C, 5% CO2. The absorbance was measured with an EL800 microplate reader  at a wavelength of 490 nm. Each experiment was conducted in triplicates and was repeated three times.The cellular viability after PDT treatment was assessed using a -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) MTS colorimetric assay (Promega CellTiter 96le cells . Followiv/v) methanol  for 10 min. Afterward, cells were washed with PBS and permeabilized with 0.2% (v/v) Triton X-100  in PBS. Nuclear staining was performed with 1% (w/v) 4′,6-diamidino-2-phenylindole  in PBS for 5 min. As a green-fluorescent cytosolic stain, 500 nm of CellTracker™ Green  was added onto the cells for 30 min. Fluorescence-stained cells were visualized with a Carl Zeiss Observer Z1 fluorescence microscope . Staurosporine  treated cells were included as a positive control  [A qualitative nuclear morphology assessment was done as previously described . Brieflyidation) . Each ex4 cells for each cell type) were subjected to a quantitative senescence-associated ß-galactosidase assay  following the manufacturer’s instructions. The absorbance was measured with a GM3510 fluorescence microplate reader  at 360 nm (Excitation)/465 nm (Emission). Three independent measurements were performed for each cell type and exposure condition.In order to investigate if PDT exposure triggers a state of stable cell cycle arrest, following PDT irradiation the seeded cells  and graphically displayed as bar diagrams. As the seeding density was adjusted for the migration assay in order to observe an effect, due to different seeding densities we refrained from performing statistical analysis. For the quantitative assays, comparison between experimental results was performed by one-way analysis of variance and t-test for independent samples as a post hoc test. Alpha adjustment based on a significance level of 0.05 was performed using the Bonferroni method. Statistical analysis was performed with SPSS Statistics 22 . As the seeding density was adjusted for the migration assay in order to observe an effect, due to different seeding densities, we refrained from performing statistical analysis.The MSC-control group showed no effect after 5-ALA PDT irradiation . In contA decrease in cell density, reduction in cytoplasm, and a smaller, altered morphology were observed for 17-1012 and MAC cells F,J,G,K wp = 0.009) and 1.5 mM (p = 0.002) at a 2000 s exposure time when compared with their corresponding controls. Contrastingly, the 17-1012 cell line exhibited an increase in viability  at an exposure of 300 s, followed by a decrease at 2000 s of exposure time (p = 0.257). The MAM cell line showed an increasing tendency in viability for both exposure times and 5-ALA concentrations . Irrespective of the exposure time and photosensitizer concentration, the number of viable MSCs remained unaltered and no notable change was observed.To investigate the effect on cellular viability after PDT exposure with 5-ALA, an MTS assay was conducted on all cell lines. Independent of the exposure time, for both of the employed 5-ALA concentrations (1 mM and 1.5 mM) a similar and comparable tendency for all of the cell lines was observed . A statiChanges in nuclear morphology as a hallmark for apoptosis were also examined qualitatively utilizing fluorescence microscopy. DAPI and cytosolic staining were used to visualize the changes in the nucleus and formation of apoptotic bodies—important hallmarks of apoptosis. It could be noted that after 2000 s of PDT exposure with 1.5 mM of 5-ALA, MAC, MAM, and 17-1012 cell lines . NuclearCellular senescence or cellular aging is characterized by a diminished replicative capacity coupled with altered functionality within the cell . To clarCurrently, radiotherapy is the gold standard in the treatment of metastases; however, it only offers a limited capacity for tissue repair and is related to severe adverse effects. Alternate approaches for treating tumors and metastases are urgently needed and one such potential candidate is the combination of photosensitizer, 5-aminolevulinic acid (5-ALA), and photodynamic therapy (PDT); however, the data on its safety and effectiveness in the treatment of metastatic lesions is limited. 5-ALA PDT offers several advantages, such as low toxicity, fast clearance, and rapid conversion to porphyrin.In this study, we assessed 5-ALA PDT treatment against different human cell lines, primary giant cell tumor of bone and bone metastases of invasive ductal carcinoma, renal carcinomas, as well as MSC (control group). We found that different human cell lines have distinct 5-ALA-PDT-sensitivities. Exposure of cells to 5-ALA PDT proved particularly effective in decreasing the migration potential, viability, cell proliferation, and density in bone metastases of invasive ductal carcinoma. For cells derived from giant cell tumor of bone, 5-ALA PDT irradiation triggered a notable decrease in migration potential, and cell density. No notable effect was observed in bone metastases of renal carcinoma and MSC group. The reduction effect, was, in general, more pronounced with a longer PDT exposure time and higher 5-ALA concentration suggesting a dose and exposure-dependent effect consistent with the previous research ,32. NumeIn fact, the cell line with lower sensitivity to 5-ALA PDT, that is, bone metastases of renal carcinoma (MAM), actually went into the state of cellular senescence caused by 5-ALA PDT exposure, and no therapeutic effect was observed. Senescence-induced cell lines showed no significant changes in cell migration even after prolonged PDT irradiation, and in fact exhibited increased viability even after a long exposure time. Thus, different sensitivity to the same doses and exposure of 5-ALA and PDT results in a completely different cell fate. While cellular senescence may be a beneficial strategy preventing the growth of the tumor short-term, cells remain metabolically active, affecting neighboring cells and stimulating cancer-promoting mechanisms ,38,39,40In bone metastases of the renal carcinoma cell line, 5-ALA PDT-induced senescence could be attributed to a too-low light dose, as the cellular decision between apoptosis/necrosis and senescence partially depends on it ,43. Our Even though nuclear morphologic changes such as chromosome condensation and fragmentation are considered hallmarks of apoptosis , DNA fraOne of the key advantages of PDT is that it can be used either before or after chemotherapy, radiotherapy, or surgery, without compromising these therapeutic modalities and the adverse effects of chemotherapy or radiation do not affect sensitivity to PDT. Several in vitro studies have shown the synergistic benefit of PDT and ionizing radiation in several pathologies ,50,51,52Human cell lines have different sensitivity to the same doses and exposure of 5-ALA PDT, resulting in two different cell fates apoptosis or senescence, depending on the extent of the cellular damage. We found that PDT has potential applicability in bone metastases of invasive ductal carcinoma. While no possible therapeutic effect was observed in bone metastases of renal carcinoma, a limited effect on primary bone tumors (giant cell bone tumor) is conceivable.While our study offers an interesting new perspective to the in vitro effects of 5-ALA PDT irradiation on metastatic cell lines, the approach implies a laborious work. For these reasons, only three independent experiments for each cell line were finally conducted. This aspect should be kept in mind when interpreting the statistical results, however, the measured tendency should not be affected. Additionally, it has to be noted that cellular heterogeneity in tumor cells is a well-established phenomenon ,54 consi


Osteosarcoma (OS) is an aggressive mesenchymal cell tumor that carries a poor long-term prognosis. Despite definitive surgery for the primary tumor and adjuvant chemotherapy, pulmonary metastasis is common and is the primary cause of morbidity. To improve outcomes for patients, we have developed and optimized a phenotypic screen for drugs that may target OS disseminated tumor cells (DTCs) and inhibit their metastatic outbreak rather than merely screening for cytotoxic activity against proliferating cells, as is commonly conducted in conventional drug discovery approaches. We report on the validation of a previously described 3D reconstituted basement membrane extract (3D BME) model system for tumor dormancy and metastatic outgrowth adapted to clonal pairs of high and low metastatic OS cells. A post-hoc validation of the assay was possible by comparing the activity of a drug in our assay with early evidence of activity in human OS clinical trials (regorafenib and saracatinib). In this validation, we found concordance between our assay and human clinical trial experience We then explored an approved veterinary small molecule inhibitor of Janus kinase-1 (oclacitinib) as a potential drug candidate to take advantage of the high prevalence of OS in pet dogs and its translational value to humans. Despite the biological rationale, we found no evidence to support the use of oclacitinib as an antimetastatic agent in OS. The findings support our 3D BME assay as a highly efficient method to examine drugs for activity in targeting OS DTCs. Osteosarcoma (OS) is a neoplasia of mesenchymal cell origin. The aggressive nature of OS results in poor prognoses, with metastasis to the lungs being the most common cause of death. Despite complete resection of the primary tumor and adjuvant chemotherapy for patients with localized disease, patients ~35%) develop recurrent metastases most commonly to the lungs within a few years and the five-year survival rate is less than 30% ,2. Accum5% develoThe current approaches to cancer drug development prioritize drugs that kill rapidly dividing cells or induce apoptosis in cancer cells. Although these targets are characteristics of cancer, they do not adequately represent the biology of DTC metastatic progression. DTCs undergo adaptation, survival, and enter a phase of dormancy and eventually outgrowth at their metastatic site. This multistep process which we coined metastatic endurance (ME)  is just Here, we describe the adaptation of a previously reported 3D reconstituted basement membrane extract (3D BME) system modeling breast cancer tumor dormancy and outgrowth ,6 to mod®), would exert a desired antimetastatic effect in 3D BME. Our hypothesis was that oclacitinib would suppress ME within OS cell lines.We first identified exposures of each drug that had minimal cytotoxic effect on two highly metastatic OS cell lines in conventional 2D conditions . We then used exposures of these drugs well below a cytotoxic threshold in the 3D BME, to identify drugs that may have selective activity against OS ME. Our primary objective was to determine if the 3D BME assay would recapitulate early human clinical trial results. Our secondary objective was to determine if oclacitinib, a currently FDA-approved veterinary drug  was active in the adapted 3D BME system, and the selected agent without measurable activity in human OS trials (saracatinib) was inactive in the adapted 3D BME system. The FDA-approved veterinary drug (oclacitinib) was not found to be active despite a hypothesized biological rationale in cancer metastasis. While our data suggests against prioritizing this drug for further development in OS, we believe the 3D BME system and other assays that model metastatic progression will uncover new therapeutics with activity against ME that are amenable to the proposed cross-species approach to metastasis drug development in OS.p < 0.005) compared to the MG63 cell line  and MG63.3 (high metastatic) cells  were culp ≤ 0.05; # p < 0.005).Similar results were found using a distinct and previously validated pair of murine OS cells, K12 (low metastatic) and K7M2 (high metastatic) cell lines . K12 celMTT assays were used to assess the influence of oclacitinib, saracatinib, and regorafenib on cell number in 2D monolayer conditions to determine the maximum physiological concentration that will not have cytotoxic activity on highly metastatic OS cells. This concentration will be further tested for inhibition of ME of highly metastatic OS cell lines in the 3D BME system. Collectively, exposures up to 1 μM were associated with minimal, consistent, and dose-dependent reductions in cell number across drugs and cell lines . Oclacitp ≤ 0.005) compared to vehicle treated cells. Significant inhibition was not seen at 100 nM. Conversely, saracatinib did not significantly inhibit the proliferative outgrowth of both cell lines in the 3D BME system at or below 1 μM. Oclacitinib also had no significant effect in the 3D BME assay on the proliferative outgrowth in either cell line tested . Two small molecular kinase inhibitors, regorafenib and saracatinib were chosen as suspected positive and negative controls respectively given their previous clinical results in early human clinical trials. The results with these two “control drugs” provide a post hoc validation of the 3D BME assay as a predictive screen for drugs with antimetastatic activity in this adaptation of the 3D BME assay. A third small kinase inhibitor was then assessed for efficacy against ME in OS cells.The experiments performed here included OS cell lines that have been rigorously evaluated for biologic and metastatic behavior ,11. The One additional challenge associated with our 3D BME assay approach is the exclusion of the innate/adaptive immune system’s influence on the metastatic niche . In lighRegorafenib, our positive control, significantly suppressed outgrowth of cells in 3D BME at clinically relevant doses, suggestive of potential antimetastatic activity. Regorafenib is already used across a multitude of cancer indications including hepatocellular carcinoma, renal cell carcinoma, and gastrointestinal stromal cell tumors ,15,16. ISaracatinib was used as a negative control for this study given its poor performance in a clinical trial in humans with progressive OS. While the clinical trial showed an increase in progression free survival there was no apparent benefit to long term overall survival . It is fOclacitinib, our drug of interest, is a Janus kinase inhibitor approved for use in controlling allergy, inflammation, and pruritus in dogs. The drug binds to JAK1/2/3 and TYK2  with itsAn additional consideration in deprioritizing oclacitinib as a therapeutic candidate is concern for its immunosuppressive properties. In initial safety studies, concern was raised about emergence of diseases commonly seen in immunosuppressed dogs  . SubsequDespite oclacitinib’s lack of efficacy reported here, we believe the described 3D BME assay approach has value as part of a phenotypic screen of cancer metastasis, that in this case could be rapidly applied to in vivo validation studies in canine OS as a model for human OS. The switch from a dormant cell to an active metastatic cell involves multiple intracellular pathways ,25 and sTwo previously described clonally related and biologically validated high and low metastatic OS cell lines MG63.3 , MG63 , K7M2 (highly metastatic murine OS), and K12 (low metastatic murine OS),  were use2.Cells were maintained and passaged in Dulbecco’s modified Eagle medium (DMEM) +4.5 g/L D-Glucose and 2 mM L-glutamine supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) , and incubated at 37 °C in 5% humidified COOclacitinib, saracatinib, and regorafenib  were each dissolved in DMSO at a stock concentration of 10 mM.The number of viable cells present in 2D culture was determined by MTT as previously reported . BrieflyThe resulting absorbance was measured at 490 nm via plate reader . DMSO (0.1%) was used as vehicle control, while 20 μM rapamycin was used as a positive cell-kill control ,28. The The influence of drug exposure on the transition from tumor dormancy to metastatic growth of OS cells was assessed in a 3D BME system as described before ,29.® PathClear, Reduced Growth Factor Basement Membrane Extract  was added (50 μL/well) to a 96-well plate and allowed to gel by incubating at 37 °C for ≥30 min. Cells were harvested, washed with DMEM , and centrifuged at 1500× g 5 min. The media was aspirated, and the remaining cell pellet flicked for ~10 s to ensure disaggregation.CultrexCells were diluted in assay media containing 2% BME, resuspended vigorously, and 150 μL of the cell suspension was added on top of the gelled 3D BME layer. For comparison, matched low-metastatic cell lines (MG63 and K12) were grown under the same conditions as the highly metastatic cells (MG63.3 and K7M2) and did not exhibit the same growth rate or kinetic profile, highlighting the differences in their metastatic potential. Proliferation was determined in the control vehicle (cells in media + 0.1% DMSO) or treated wells at indicated days by the addition of 20 μL/well of MTS reagent , followed by incubation at 37 °C for 2 h and absorbance measurements at 490 nm via plate reader .All conditions were conducted in quadruplicate and each experiment was carried out at least 3 separate times. All comparisons were made between treatment and vehicle control at concordant time points. Statistical analyses and non-linear regressions were derived using GraphPad Prism statistical software.The results reported here validate a modified 3D BME assay system for use with OS cells to assess efficacy of drugs or therapeutic agents against ME satisfying our primary objective. We showed that the assay system was effective at mirroring previously known clinical results with two drugs: regorafenib and saracatinib. In 3D BME, regorafenib suppressed metastatic outgrowth of two highly metastatic OS cell lines while saracatinib was ineffective. Oclacitinib also proved ineffective at suppressing ME in two highly metastatic cell lines, refuting our working hypothesis. Next steps include expansion of the number of cell lines used in 3D BME testing, and continued examination of drugs with potential for translation to anti-metastatic agents. Once identified, these agents will be further evaluated for efficacy against ME using the ex vivo pulmonary metastasis assay to better model an in vivo metastatic niche. Drugs found to show continued efficacy against ME in the PuMA may be brought forward for clinical trials, specifically trials in the canine OS population as a model for human OS.


Colorectal cancer (CRC) is the fourth most lethal disease worldwide. Despite an urgent need for therapeutic advance, selective target identification in a preclinical phase is hampered by molecular and metabolic variations between cellular models. To foster optimal model selection from a translational perspective, we performed untargeted ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry-based polar metabolomics and lipidomics to non-transformed (CCD841-CON and FHC) and transformed  colon cell lines as well as tissue samples from ten colorectal cancer patients. This unveiled metabolic signatures discriminating the transformed from the non-transformed state. Metabolites involved in glutaminolysis, tryptophan catabolism, pyrimidine, lipid and carnitine synthesis were elevated in transformed cells and cancerous tissue, whereas those involved in the glycerol-3-phosphate shuttle, urea cycle and redox reactions were lowered. The degree of glutaminolysis and lipid synthesis was specific to the colon cancer cell line at hand. Thus, our study exposed pathways that are specifically associated with the transformation state and revealed differences between colon cancer cell lines that should be considered when targeting cancer-associated pathways. Although the discovery of CRC-biomarkers and associated pathways has contributed to enhanced screening and improved treatment, the 5-year survival rate of metastatic CRC is still only 10%, emphasizing the need for better therapeutic strategies3.Colorectal cancer (CRC) is the second and third most diagnosed cancer in females and males, respectively, and the fourth leading cause of cancer-related mortality worldwide. The incidence rates are strongly variable throughout the world, whereby developed regions have more CRC patients than less developed countries. Disease risk is primarily associated with lifestyle factors, in particular Western habits such as the consumption of diets rich in fat and sugar, high in red and processed meat and limited physical activitye.g., azoxymethane) or genetically  induced cancer rodent models6. In keeping with the 3R principles , a framework that aims at minimizing animal use and suffering, researchers continuously seek bona fide alternatives wherever possible7. Therefore, many in vitro models, especially cell culture models, have been established to study CRC and therapeutic strategies8. Evidently, cell lines have less ethical constraints in comparison to animal models, and at the same time they are much easier in use, inexpensive and provide sufficient material, enabling rapid experimental progress9.In cancer research, animal models have been of great value for discovering novel biochemical pathways and drug testing. The most common models are xenograft and chemically  cell lines are both immortalized, but non-transformed (NT) colon cell lines obtained from fetal normal colon mucosa13. On the other side of the spectrum, transformed (T) cell lines isolated from (adeno)carcinomas may serve as representative samples for studying different cancer stages. For example, HT29, SW480 and SW948 were established from colorectal adenocarcinomas, whilst Caco2 and HCT116 were established from colorectal carcinomas14. These cell lines have been extensively used to study regulatory mechanisms in CRC as well as for the purpose of identifying chemopreventive and -therapeutic agents15. Preclinical data using in vivo and in vitro models revealed that only 5% of candidate therapies demonstrate clinical efficacy in phase III trials. This high attrition rate can be attributed to some extent to the use of models that are not fully representative for the type of cancer of interest16. For example, it has been demonstrated that many cancer cell lines used to model various types of cancers were in fact derived from the HeLa cervical cancer cell line and not the corresponding tumor17. Moreover, it was also observed that long-term passaging of cells can lead to a reduced resemblance to the tumor of origin14. Therefore, several efforts have been made to reduce cell line misidentification, primarily by molecular phenotyping19. Although metabolites are the end-products in the gene-protein-metabolite cascade and thus key reporters of cellular activity, metabolic profiling or fingerprinting studies of cancer cells and tissue are scarce20. Pioneering studies have revealed a conspicuous metabolic rewiring in CRC. For example, both CRC tissue and cell lines have been found to display higher levels of phospholipids, of which certain  correlate with metastatic propensity22. Also, specific nucleotides and carbohydrates have been found upregulated in CRC patient tissue23. The latter may represent metabolic evidence for a Warburg phenotype21, although higher rates of oxidative phosphorylation have been reported in CRC cell lines as well23. Thus, our understanding of the exact metabolic status of CRC cells is far from complete. And, to the best of our knowledge, no studies are available that assessed whether metabolic differences between healthy and cancerous colon tissue can be extrapolated to cell cultures and vice versa. We have previously optimized and validated an untargeted ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry-based (UHPLC-HRMS) metabolomics and lipidomics method for human colon tissue and colon cell lines24. In this work, we exploited the approach to unveil pathways linked to the CRC transformation processes in vivo and in vitro and to provide novel insights into the metabolic phenotype of different cell lines that could foster the selection of the optimal one(s) towards specific targets in cancer drug research.Whilst primary cell cultures from healthy patients may be considered to resemble the native state more closely, they are less practical to culture and have limited life span. Immortalized cell lines make a valuable alternative since they are still representative for the tissue of origin and are easier to handle and to maintain in culture25, all cell lines of one passage were harvested at the same day, extracted and analyzed for metabolomics directly after. The remaining cell pellets were stored at − 80 °C and subjected for joint lipidomic extraction and analysis . One sample of the FHC line (P3) was excluded for having too limited starting material. Chromatographic peak processing resulted in the detection of in total 879 metabolite and 17,432 lipid components, after removing those with a coefficient of variation (CV) > 30% in quality control (QC) samples composed of cell line or tissue matrix extracts. Valid orthogonal partial least squares discriminant analysis (OPLS-DA) models , which implies they had a significant contribution to the resulting model (as indicated by a Variable Importance parameter (VIP) value > 1.0). Thus, the untargeted UHPLC-HRMS approach revealed a specific set of metabolomic components that typify the transformation state (NT vs T) of cells and tissue.Untargeted UHPLC-HRMS metabolomics and lipidomics profiling was performed on samples of two NT (FHC and CCD 841 CON) and five T  colon cell lines as well as on cancerous (T) and flanking non-cancerous (NT) tissue biopsy samples from 10 CRC patients. Of each cell line, cellular extracts were obtained from three culture flasks  at three different passages (P1-3), separated by one-week intervals . To avoid metabolic shifts during storage from introducing bias between cell lines, whereof 35 and 52 were discriminative  for the transformation state in colon tissue and cell lines, respectively  for colon tissue and cell lines confirmed their potential to discriminate transformation states and differentiate HT29 and HCT116 from SW480 and HCT116 cell lines (Table The accurate mass of 1626 discriminative components was queried in the HMDB and LIPIDMAPS for putative identifiers (IDs). 377 IDs could be linked to components that were discriminative for the NT or T state in colon cell lines or tissue and were subsequently subjected to fragmentation experiments. This resulted in the identification of 55 metabolites with the highest levels of confidenceP value < 0.05) different between the cancer cell lines  affected pathways for the colon cell line and tissue matrix, respectively  lower in the SW480 than in the HCT116 cell line samples, implying more conversion of L-glutamine to L-glutamic acid in the SW480 cell line. The HT29 and Caco2 cell lines demonstrated similar conversion rates, whilst the ratio was less consistent in the SW948 cell line, as indicated by the high standard deviation  and the SW480 cell line , aligning with the higher L-glutamine/L-glutamic acid ratio and the lower conversion of L-glutamine to L-glutamic acid in the HCT116 cell line , who observed higher L-glutamic acid and lower L-glutamine levels in colon tumor tissue, respectively26. Amongst the T cell lines, the SW480 cell line showed the highest rate of glutaminolysis as represented by higher conversion of L-glutamine towards L-glutamic acid. Also, qPCR results showed higher expression of glutaminase in this cell line indicating more enzyme availability promoting this metabolite conversion. Compared to HCT116 cells, SW480 cells displayed significantly lower L-glutamine and L-glutamic acid levels, which could indicate lower absorption of L-glutamine, but higher efficiency in utilizing this metabolite as an energy source for the latter. Interestingly, it has been shown that the removal of L-glutamine from media results in increased sensitivity of the SW480 as opposed to HCT116 and HT29 cell cultures, hence supporting the function of this metabolite as a more convenient energy source in the former27. L-leucine was more abundant in this cell line compared to the other cancer cell lines, which may hint towards increased activation of glutamate hydrogenase. Glutamate hydrogenase converts L-glutamic acid into α-ketoglutaric acid, an intermediate of the TCA cycle, thus promoting glutaminolytic activity28. Targeting glutaminolysis by inhibiting involved enzymes in cancer has gained interest and has already been proven to reduce the proliferation of cancer cells29.L-glutamic acid was characteristic for colon cancer cell lines and tumor tissue and is generated during glutaminolysis, 30. This is also in concordance with the higher presence of pyrimidines  and lipids (mainly PCs) in T samples31. In our study, especially saturated, mono-unsaturated and low-polyunsaturated PCs were discriminative for transformation, which aligns with a study that performed lipidomics on breast cancer and normal tissue and cell line samples32. Especially the HT29 and Caco2 cell line displayed relatively high PC levels. The HCT116 cell line was characterized by a lower abundance of lipid species, which may be linked to its decreased metastatic potential. Carcinogenesis has been linked with de novo lipogenesis and increased saturation of membrane lipids. The latter results in decreased membrane fluidity and permeability, which make cancer cells less prone to lipid peroxidation and chemotherapy in comparison to normal cells22. Based on this, it can be speculated that strategies to modulate membrane fluidity could improve the treatment of cancer33. Glycerol-3-phosphate, an intermediate in lipid synthesis, was detected at lower concentrations in the T state as opposed to the NT state, which further supports increased phospholipid biosynthesis in the T state34.Only recently, it has been shown that carbon and nitrogen of L-glutamine is incorporated into building blocks of cells, especially during high cell proliferation37. It is a common feature that ROS are elevated in cancer cells, where they are responsible for activating signaling pathways involved in cell proliferation38. However, when ROS are present in excessive amounts, they cause oxidative damage to macromolecules. To prevent this, cancer cells overproduce antioxidants30. Indeed, Kibi et al. (2019) observed higher taurine levels in CRC as opposed to adjacent tissue samples derived from 10 patients39. Therefore, one would intuitively expect that increased antioxidants would be linked to the T state, which was not the case in our work. Nevertheless, Hernandez-Lopez (2018) demonstrated that antioxidant responses between non-tumor adjacent tissue samples retrieved from patients with different stages of CRC differed, where non-tumor adjacent tissue of stage IV showed increased antioxidant levels in comparison to those of stage III40. In our work, 3 CRC patients had classification of tumors below stage II, whereas the tumors of the other 7 patients were classified in stage III and IV  were determined to be discriminative for NT samples in vitro and in vivo and are considered to be important in mitigating the damaging effects of ROS. These compounds act as a coenzyme in redox reactions, improve mitochondrial function resulting in less superoxide generation and scavenge superoxide radicals, respectivelyet al. 209 observe41.The urea cycle is a metabolic pathway that is responsible for converting excess nitrogen from L-aspartic acid and ammonia, which results from amino acid catabolism, into urea. The urea cycle metabolites L-arginine and citrulline were downregulated in T cell lines as opposed to NT cell lines. These results are in accordance with Nagamani et al. (2016), who demonstrated that metabolic wiring accompanying carcinogenesis includes enhanced flux of L-aspartic acid towards pyrimidine synthesis and hence, decreased synthesis of L-arginine42. L-kynurenine, which was elevated in the T state in colon tissue and cell lines, is associated with tumor immune escape and growth and hence, is considered as a hallmark of carcinogenesis43.Tryptophan metabolism involves the conversion of L-tryptophan into N’-formylkynurenine and L-kynurenine and this is depending primarily on indoleamine-2,3-oxygenase (IDO1), which is highly expressed in the colon. IDO-1 is one of the most upregulated in CRC and is strongly induced by inflammatory cytokines such as IFNγ38. L-lysine is a precursor of N6,N6,N6-trimethyllysine, which can be converted to L-carnitine and further transformed to L-ascorbic acid. L-Carnitine is responsible for transporting fatty acids into the mitochondria or peroxisomes by conjugation with the acyl-CoA moiety to form acylcarnitines. Inside these cell organelles, acyl-CoA is released and enters the β-oxidation resulting in the formation of acetyl-CoA, that can in turn enter the TCA cycle37. In this study, only L-carnitine showed a clear association with the T state in both matrices. L-lysine, L-ascorbic acid and N6,N6,N6-trimethyllysine on the other hand were significantly elevated in the NT as opposed to the T state in cell line samples, but showed an opposite trend in the T colon tissue samples. Nevertheless, this observation was not significant for L-lysine and N6,N6,N6-trimethyllysine. Based on these findings, it can be concluded that L-carnitine biosynthesis and lysine degradation are higher in the T state. However, at the tissue level, such metabolic fluxes were less clearly observed, which can be due to genetic heterogeneity of samples. In concordance with these findings, elevated carnitine levels in plasma and tissue have been associated with breast, bladder and colon cancer, respectively46.It has been reported that L-carnitine is elevated in cancer cells and associated with enhanced fatty acid uptake, oxidation and formation of ROS47. Conversely, cells in tumor tissue are subjected to harsh conditions, including hypoxia and limited nutrient availability48. In this study, it was observed that L-ascorbic acid was significantly upregulated in the T as opposed to the NT tissue samples while a higher abundance was detected in NT as compared to the T cell line samples. In vivo trials have demonstrated that L-ascorbic acid is more abundant in cancer cells than normal cells through facilitated transport by glucose transporters, explaining the higher concentrations in colon cancer tissue. Nevertheless, this metabolite acts as a pro-oxidant in tumor cells, since these lack the enzyme catalase that converts L-ascorbic acid-induced ROS (H2O2) into water and oxygen49. Thus, cancer cells are more vulnerable than normal cells to the effects of this metabolite and can eventually stop growing or undergo apoptosis51. Hence, it could be hypothesized that cell lines have adapted to culture conditions by rewiring their utilized energy sources, for example through the absorption and use of L-glutamine, which is highly abundant in commercial cell culture media. This way, cancer cell lines are more protected against the detrimental effects of L-ascorbic acid. Indeed, recent research has shown that increased extracellular availability of glucose does not influence the rate of glucose utilization by cell line models. It was also demonstrated that higher glutamine concentrations in media resulted in higher glutamine fermentation in cell culture52.It is important to note that discrepancies were observed in metabolic profiles between the cell line and tissue samples. This may be due to the cell culture methodology that differs from the in vivo situation. For example, cell lines do not have a physiological extracellular matrix, 3D organization, and are much more homogeneous than tissue. Also, cell lines are adapted to their culture conditions, whereby media are composed in such a way that cell growth and viability are optimally supportedIn conclusion, despite the fact that in vitro cell culture models do not fully represent the in vivo situation, we discovered shared metabolites and corresponding pathways between colon tissue and cell lines involved in carcinogenesis. Glutaminolysis, associated pyrimidine and lipid synthesis, tryptophan catabolism, glycerol-3-phosphate shuttle and carnitine biosynthesis were enhanced in the malignant as opposed to the non-malignant state. In the NT state, urea cycle and antioxidant levels were more prominent. Moreover, it was clear that glutaminolysis, characterized by high expression of glutaminase and high conversion of L-glutamine to L-glutamic acid, was most abundant in the SW480 cell line as opposed to the other T cell lines. In addition, the HT29 and Caco2 cell lines contained the most saturated and one or double bonds-containing phospholipids, essential constituents of cell membranes and thus affecting membrane permeability towards cancer drugs. Hence, this work provides insights in pathways associated with colon carcinogenesis as well as valuable information to select the most appropriate cancer cell lines in the testing of novel chemotherapeutic agents.2/95% air. FHC cells were cultured in DMEM:F12 supplemented with 10 mM HEPES, 0.005 mg mL-1 insulin, 0.005 mg mL-1 transferrin, 100 ng mL-1 hydrocortison, 10% FBS and 1% P/S. All cell reagents were purchased from Life Technologies .The human colorectal cell lines HT29, Caco2, HCT116, SW480 and SW948 (T state) and the immortalized colon cell lines FHC and CCD841-CON (NT state) were obtained from ATCC . Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin (P/S), was used to culture the cancer cell lines and CCD841-CON in a humidified incubator at 37 °C and 5% COCancerous colon material (100 mg) and corresponding healthy tissue (100 mg) from 10 individuals were provided by Biobank@UZA . The use of human colon tissue samples in this study was ethically approved by the University Hospital of Antwerp  and patient details are listed in Supplementary Table . (2019)24. For each matrix , quality control (QC) samples were made from a pool of the matrix-specific samples and run in duplicate after every 10 samples.In recent work, the applied two-step metabolomics and lipidomics extraction protocols for colon (cancer) cell lines and tissue were successfully optimized and validated. More information about the optimized parameters and validation experiments can be consulted in Rombouts et al54. Solvents were of LC–MS grade for extraction purposes and obtained from Fisher Scientific  and VWR International . Ultrapure water was obtained by usage of a purified-water system .Analytical standards were purchased from Sigma-Aldrich , ICN Biomedicals Inc. , TLC PharmChem  or Cambridge Isotope Laboratories Inc. . Detailed information about chromatographic and mass spectrometric features of the reference metabolite and lipid compounds can be consulted in De Paepe et al. (2018) and Van Meulebroek et al. (2017), respectively53. For fragmentation experiments, the following MS/MS settings were applied during parallel reaction monitoring (PRM): resolution of 17,500 Full Width at Half maximum (FWHM), Automatic Gain Control (AGC) target of 2 × e4 ions, maximum injection time of 40 ms and an isolation window of 2.0 m/z.An Ultimate 3000 XRS UHPLC system  and a Q-Exactive™ stand-alone bench top Orbitrap mass spectrometer (MS) , equipped with a heated electrospray ionization source (HESI II) operating in polarity switching mode, were used for polar metabolomics and lipidomics analysis as described by De Paepe et al. (2018) and Van Meulebroek et al. (2017), respectively24. In brief, components were identified using the following criteria: a minimum peak intensity of 500,000 au, minimum signal-to-noise ratio of 10, minimum number of isotopes of 2, retention time window of 0.75 min, maximum retention time shift of 0.25 min and maximum mass shift at 5 ppm. This resulted in the construction of one metabolomics dataset (two ionization modes combined) and two lipidomics datasets (positive and negative ionization modes separately). Next, unsupervised hierarchical clustering  and multivariate statistical analysis  were performed, whereby OPLS-DA models were built to discriminate the NT from the T state in tissue and cell lines separately. For modeling, data were log-transformed and Pareto-scaling was applied55. Validity of the obtained OPLS-DA models was evaluated based on several parameters including R2Y (> 0.5), Q2 (> 0.5), CV-ANOVA (p < 0.05), as well as permutation testing (n = 100)56. Selection of components that discriminated between the NT and T state in cell lines and tissue was based on a Variable Importance in Projection (VIP) score > 1.0 and a Jack Knife confidence interval that did not include 057. Screening of components of interest was established based on accurate mass (Δ ppm < 6 ppm) in the Human Metabolome Database (HMDB) and LIPIDMAPS59. For those components that could be linked to putative IDs, fragmentation experiments were conducted, and MS/MS profiles were scored in CSI FingerID or compared with these of in-house analytical standards60. Components were then classified into different levels of assignment according to the recommendations for standard metabolite identification from the Chemical Analysis Working Group61. Based on this, retained compounds were described as identified compound  or putatively annotated compound .All cellular LC–MS data, together with tissue LC–MS data were processed simultaneously using Compound Discoverer™ 2.1 or Sieve™ 2.2 software  for the metabolomics and lipidomics experiment, respectively. Herein, chromatographic peak alignment and component extraction was performed as described earlierP value  < 0.05) in MetaboAnalyst 4.0, thereby using the independent two-sample or paired sample Wilcoxon test for, data from the colon cell lines and tissue samples, respectively. To this end, metabolites obtained from the tissue matrix were also statistically evaluated in the cell line matrix and vice versa. This approach allowed pinpointing metabolites relevant for discrimination between NT and T state in tissue or cell lines as well as those relevant in both matrices making extrapolation and comparison easier. Significantly different metabolites between the transformed cell line types were assessed using One-Way Anova and Tukey-adjusted post hoc testing for all pairwise comparisons, whereby P value s < 0.05 were considered significant.Identified and putatively annotated metabolites were statistically evaluated  were constructed and hierarchical clustering (Pearson correlation coefficient and Ward linkage) was performed of the colon cell line and tissue samples in MetaboAnalyst 4.0 . This software program was also used for quantitative enrichment analysis using biologically relevant human metabolic pathways of the Small Molecular Pathway Database (SMDB)62. Pathways with a false discovery rate (FRD) < 0.05 were considered significantly altered. Next to this, the KEGG Compound Database (www.genome.jp/kegg/compound) and scientific literature was consulted to provide a broader overview of transformation-associated pathways in colon tissue and cell lines.Heat maps presenting the abundances of the discriminative metabolites  according to the manufacturer’s instructions. Primers were used for GLS1  and reference genes GADPH , ACTB  and TUB 63. qPCR technical replicates (n = 4) that deviated more than 0.3 Cq units and genes with inconsistent melting curves were removed from downstream analysis. Relative quantification was performed using the 2−ΔΔCT method, whereby the HT29 samples were used as reference64. One-way ANOVA together with Tukey-adjusted post hoc testing for all pairwise comparisons was performed in RStudio 2.1, whereby P value s < 0.05 were considered significant.RNA was extracted using the Nucleospin Triprep kit  and quality control was performed with the NanoDrop 2000 . Next, 1 μg of RNA was converted to cDNA synthesis with Superscript® II Reverse Transcriptase (RT)  . qPCR reactions were performed on a CFX Connect™ system  using SsoAdvanced™ Universal SYBRSupplementary Information.