NMR-Based Metabolomics of Blood Serum in Predicting Response to Induction Chemotherapy in Head and Neck Cancer-A Preliminary Approach.

The role of induction chemotherapy (iCHT) in locally advanced head and neck squamous cell carcinoma (LA-HNSCC) is still to be established due to high toxicity and variable response rates. The aim of this retrospective study is to use NMR-based serum metabolomics to predict the response rates to iCHT from the pretreatment samples. The studied group consisted of 46 LA-HNSCC patients treated with iCHT. The response to the treatment was evaluated by the clinical, fiberoptic, and radiological examinations made before and after iCHT. The proton nuclear magnetic resonance (1H NMR) serum spectra of the samples collected before iCHT were acquired with a 400 MHz spectrometer and were analyzed using multivariate and univariate statistical methods. A significant multivariate model was obtained only for the male patients. The treatment-responsive men with >75% primary tumor regression after iCHT showed pretreatment elevated levels of isoleucine, alanine, glycine, tyrosine, N-acetylcysteine, and the lipid compounds, as well as decreased levels of acetate, glutamate, formate, and ketone bodies compared to those who did not respond (regression of the primary tumor <75%). The results indicate that the nutritional status, capacity of the immune system, and the efficiency of metabolism related to protein synthesis may be prognostic factors for the response to induction chemotherapy in male HNSCC patients. However, larger studies are required that would validate the findings and could contribute to the development of more personalized treatment protocols for HNSCC patients.

X-ray-Sensitive Doped CaF2-Based MRI Contrast Agents for Local Radiation Dose Measurement.

Ionizing radiation has become widely used in medicine, with application in diagnostic techniques, such as computed tomography (CT) and radiation therapy (RT), where X-rays are used to diagnose and treat tumors. The X-rays used in CT and, in particular, in RT can have harmful side effects; hence, an accurate determination of the delivered radiation dose is of utmost importance to minimize any damage to healthy tissues. For this, medical specialists mostly rely on theoretical predictions of the delivered dose or external measurements of the dose. To extend the practical use of ionizing radiation-based medical techniques, such as magnetic resonance imaging (MRI)-guided RT, a more precise measurement of the internal radiation dose internally is required. In this work, a novel approach is presented to measure dose in liquids for potential future in vivo applications. The strategy relies on MRI contrast agents (CAs) that provide a dose-sensitive signal. The demonstrated materials are (citrate-capped) CaF2 nanoparticles (NPs) doped with Eu3+ or Fe2+/Fe3+ ions. Free electrons generated by ionizing radiation allow the reduction of Eu3+, which produces a very small contrast in MRI, to Eu2+, which induces a strong contrast. Oxidative species generated by high-energy X-rays can be measured indirectly using Fe2+ because it oxidizes to Fe3+, increasing the contrast in MRI. Notably, in the results, a strong increase in the proton relaxation rates is observed for the Eu3+-doped NPs at 40 kV. At 6 MV, a significant increase in proton relaxation rates is observed using CaF2 NPs doped with Fe2+/Fe3+ after irradiation. The presented concept shows great promise for use in the clinic to measure in vivo local ionizing radiation dose, as these CAs can be intravenously injected in a saline solution.

Metabolite Biomarkers of Prolonged and Intensified Pain and Distress in Head and Neck Cancer Patients Undergoing Radio- or Chemoradiotherapy by Means of NMR-Based Metabolomics-A Preliminary Study.

Treatment of head and neck squamous cell carcinoma (HNSCC) has a detrimental impact on patient quality of life. The rate of recognized distress/depression among HNSCC patients ranges from 9.8% to 83.8%, and the estimated prevalence of depression among patients receiving radiotherapy is 63%. Shorter overall survival also occurs in preexisting depression or depressive conditions. The present study analyzes the nuclear magnetic resonance (NMR) blood serum metabolic profiles during radio-/chemoradiotherapy and correlates the detected alterations with pain and/or distress accumulated with the disease and its treatment. NMR spectra were acquired on a Bruker 400 MHz spectrometer and analyzed using multivariate methods. The results indicate that distress and/or pain primarily affect the serum lipids and metabolites of energy (glutamine, glucose, lactate, acetate) and one-carbon (glycine, choline, betaine, methanol, threonine, serine, histidine, formate) metabolism. Sparse disturbances in the branched-chain amino acids (BCAA) and in the metabolites involved in protein metabolism (lysine, tyrosine, phenylalanine) are also observed. Depending on the treatment modality-radiotherapy or concurrent chemoradiotherapy-there are some differences in the altered metabolites.

The Relationship between Histological Composition and Metabolic Profile in Breast Tumors and Peritumoral Tissue Determined with 1H HR-MAS NMR Spectroscopy.

Breast tumors constitute the complex entities composed of cancer cells and stromal components. The compositional heterogeneity should be taken into account in bulk tissue metabolomics studies. The aim of this work was to find the relation between the histological content and 1H HR-MAS (high-resolution magic angle spinning nuclear magnetic resonance) metabolic profiles of the tissue samples excised from the breast tumors and the peritumoral areas in 39 patients diagnosed with invasive breast carcinoma. The total number of the histologically verified specimens was 140. The classification accuracy of the OPLS-DA (Orthogonal Partial Least Squares Discriminant Analysis) model differentiating the cancerous from non-involved samples was 87% (sensitivity of 72.2%, specificity of 92.3%). The metabolic contents of the epithelial and stromal compartments were determined from a linear regression analysis of the levels of the evaluated compounds against the cancer cell fraction in 39 samples composed mainly of cancer cells and intratumoral fibrosis. The correlation coefficients between the levels of several metabolites and a tumor purity were found to be dependent on the tumor grade (I vs II/III). The comparison of the levels of the metabolites in the intratumoral fibrosis (obtained from the extrapolation of the regression lines to 0% cancer content) to those levels in the fibrous connective tissue beyond the tumors revealed a profound metabolic reprogramming in the former tissue. The joint analysis of the metabolic profiles of the stromal and epithelial compartments in the breast tumors contributes to the increased understanding of breast cancer biology.

Metabolomic Insight into Implications of Induction Chemotherapy Followed by Concomitant Chemoradiotherapy in Locally Advanced Head and Neck Cancer.

The present study compares two groups of locally advanced patients with head and neck squamous cell carcinoma (LA-HNSCC) undergoing concurrent chemoradiotherapy (cCHRT), specifically those for whom it is a first-line treatment and those who have previously received induction chemotherapy (iCHT). The crucial question is whether iCHT is a serious burden during subsequent treatment for LA-HNSCC and how iCHT affects the tolerance to cCHRT. Of the 107 LA-HNSCC patients, 54 received cisplatin-based iCHT prior to cCHRT. The patients were clinically monitored at weekly intervals from the day before until the completion of the cCHRT. The 843 blood samples were collected and divided into two aliquots: for laboratory blood tests and for nuclear magnetic resonance (NMR) spectroscopy (a Bruker 400 MHz spectrometer). The NMR metabolites and the clinical parameters from the laboratory blood tests were analyzed using orthogonal partial least squares analysis (OPLS) and the Mann-Whitney U test (MWU). After iCHT, the patients begin cCHRT with significantly (MWU p-value < 0.05) elevated blood serum lipids, betaine, glycine, phosphocholine, and reticulocyte count, as well as significantly lowered NMR inflammatory markers, serine, hematocrit, neutrophile, monocyte, red blood cells, hemoglobin, and CRP. During cCHRT, a significant increase in albumin and psychological distress was observed, as well as a significant decrease in platelet, N-acetyl-cysteine, tyrosine, and phenylalanine, in patients who received iCHT. Importantly, all clinical symptoms (except the decreased platelets) and most metabolic alterations (except for betaine, serine, tyrosine, glucose, and phosphocholine) resolve until the completion of cCHRT. In conclusion, iCHT results in hematological toxicity, altered lipids, and one-carbon metabolism, as well as downregulated inflammation, as observed at the beginning and during cCHRT. However, these complications are temporary, and most of them resolve at the end of the treatment. This suggests that iCHT prior to cCHRT does not pose a significant burden and should be considered as a safe treatment option for LA-HNSCC.

Grading of endometrial cancer using 1H HR-MAS NMR-based metabolomics.

The tissue metabolomic characteristics associated with endometrial cancer (EC) at different grades were studied using high resolution (400 MHz) magic angle spinning (HR-MAS) proton spectroscopy. The metabolic profiles were obtained from 64 patients (14 with grade 1 (G1), 33 with grade 2 (G2) and 17 with grade 3 (G3) tumors) and compared with the profile acquired from 10 patients with the benign disorders. OPLS-DA revealed increased valine, isoleucine, leucine, hypotaurine, serine, lysine, ethanolamine, choline and decreased creatine, creatinine, glutathione, ascorbate, glutamate, phosphoethanolamine and scyllo-inositol in all EC grades in reference to the non-transformed tissue. The increased levels of taurine was additionally detected in the G1 and G2 tumors in comparison to the control tissue, while the elevated glycine, N-acetyl compound and lactate-in the G1 and G3 tumors. The metabolic features typical for the G1 tumors are the increased dimethyl sulfone, phosphocholine, and decreased glycerophosphocholine and glutamine levels, while the decreased myo-inositol level is characteristic for the G2 and G3 tumors. The elevated 3-hydroxybutyrate, alanine and betaine levels were observed in the G3 tumors. The differences between the grade G1 and G3 malignances were mainly related to the perturbations of phosphoethanolamine and phosphocholine biosynthesis, inositol, betaine, serine and glycine metabolism. The statistical significance of the OPLS-DA modeling was also verified by an univariate analysis. HR-MAS NMR based metabolomics provides an useful insight into the metabolic reprogramming in endometrial cancer.

NMR-Based Metabolomics in Investigation of the Radiation Induced Changes in Blood Serum of Head and Neck Cancer Patients and Its Correlation with the Tissue Volumes Exposed to the Particulate Doses.

In the present study, we analyze the nuclear magnetic resonance (NMR) blood serum metabolic profiles of 106 head and neck squamous cell carcinoma (HNSCC) patients during radio (RT) and concurrent radio-chemotherapy (CHRT). Four different fractionation schemes were compared. The blood samples were collected weekly, from the day before the treatment until the last week of CHRT/RT. The NMR spectra were acquired on A Bruker 400 MHz spectrometer at 310 K and analyzed using multivariate methods. Seven metabolites were found significantly to be altered solely by radiotherapy: N-acetyl-glycoprotein (NAG), N-acetylcysteine, glycerol, glycolate and the lipids at 0.9, 1.3 and 3.2 ppm. The NMR results were correlated with the tissue volumes receiving a particular dose of radiation. The influence of the irradiated volume on the metabolic profile is weak and mainly limited to sparse correlations with the inflammatory markers, creatinine and the lymphocyte count in RT and the branched-chain amino-acids in CHRT. This is probably due to the optimal planning and delivery of radiotherapy improving sparing of the surrounding normal tissues and minimizing the differences between the patients (caused by the tumor location and size).

Molecular response to induction chemotherapy and its correlation with treatment outcome in head and neck cancer patients by means of NMR-based metabolomics.

BACKGROUND: The aim of this prospective study is to identify the biomarkers associated with the effects of induction chemotherapy (iCHT) in terms of the favorable/weaker response to the treatment in locally advanced head and neck squamous cells carcinomas (LA-HNSCC). METHODS: The studied group consisted of 53 LA-HNSCC patients treated with iCHT. The treatment tolerance was measured by the Common Terminology Criteria for Adverse Events (CTCAE). The response to the treatment was evaluated by the clinical, fiberoptic and radiological examinations made before and after iCHT (the TNM Classification of Malignant Tumors was used for classifying the extent of cancer spread). Proton nuclear magnetic resonance (1H NMR) serum spectra of the samples collected before and after iCHT were acquired with a 400 MHz spectrometer and analyzed using the multivariate and univariate statistical methods. RESULTS: The molecular response to iCHT involves an increase of the serum lipids which is accompanied by the simultaneous decrease of alanine, glucose and N-acetyl-glycoprotein (NAG). Furthermore, in males, the iCHT induced changes in the lipid signals and NAG significantly correlate with the regression of the primary tumor. The OPLS-DA multivariate model identified two subgroups of the patients with a weaker metabolic and clinical response. The first one consisted of the patients with a significantly lower initial nodal stage, the second one showed no differences in the initial clinical and metabolic statuses. CONCLUSIONS: The NMR-based metabolomic study of the serum spectra revealed that iCHT induces the marked changes in the LA-HNSCC patients' metabolic profiles and makes it possible to stratify the patients according to their response to iCHT. These effects are sex dependent. Further studies on a larger scale accounting for sex and the clinical and metabolic factors are warranted.

Shared and unique metabolic features of the malignant and benign thyroid lesions determined with use of 1H HR MAS NMR spectroscopy.

The purpose of this work was to investigate the distinct and common metabolic features of the malignant and benign thyroid lesions in reference to the non-transformed tissue from the contralateral gland (chronic thyroiditis and colloid goiter). 1H HR MAS NMR spectra of 38 malignant lesions, 32 benign lesions and 112 samples from the non-tumoral tissue (32 from chronic thyroiditis and 80 samples from colloid goiter) were subjected both to multivariate and univariate analysis. The increased succinate, glutamine, glutathione, serine/cysteine, ascorbate, lactate, taurine, threonine, glycine, phosphocholine/glycerophosphocholine and decreased lipids were found in both lesion types in comparison to either colloid goiter or chronic thyroiditis. The elevated glutamate and choline, and reduced citrate and glucose were additionally evident in these lesions in reference to goiter, while the increased myo-inositol-in comparison to thyroiditis. The malignant lesions were characterized by the higher alanine and lysine levels than colloid goiter and thyroiditis, while scyllo-inositol was uniquely increased in the benign lesions (not in cancer) in comparison to both non-tumoral tissue types. Moreover, the benign lesions presented with the unique increase of choline in reference to thyroiditis (not observed in the cancerous tissue). The metabolic heterogeneity of the non-tumoral tissue should be considered in the analysis of metabolic reprogramming in the thyroid lesions.

Monitoring of diffusion properties and transverse relaxation time of mouse ischaemic muscle after administration of human mesenchymal stromal cells derived from adipose tissue.

OBJECTIVES: Application of non-invasive imaging methods plays an important role in the assessment of cellular therapy effects in peripheral artery disease. The purpose of this work was to evaluate the kinetics of MRI-derived parameters characterizing ischaemic hindlimb muscle after administration of human mesenchymal stromal cells derived from adipose tissue (hADSC) in mice. MATERIALS AND METHODS: MRI experiments were performed on a 9.4T Bruker system. The measurement protocol included transverse relaxation time mapping and diffusion tensor imaging. The monitoring period encompassed 14 days after femoral artery ligation and subsequent cell administration. The effect of hADSC transplantation was compared with the effect of normal human dermal fibroblasts (NHDFs) and phosphate-buffered saline injection. RESULTS: The most significant differences between the hADSC group and the remaining ones were observed around day 3 after ischaemia induction (increased transverse relaxation time in the hADSC group in comparison with the control group) and around day 7 (increased transverse relaxation time and decreased third eigenvalue of the diffusion tensor in the hADSC group in comparison with the control and NHDF groups) at the site of hADSC injection. Histologically, it was associated with increased macrophage infiltration at days 3-7 and with the presence of small regenerating fibres in the ischaemic tissue at day 7. CONCLUSIONS: Our results underscore the important role of macrophages in mediating the therapeutic effects of hADSCs and confirm the huge potential of magnetic resonance imaging in monitoring of cellular therapy effects.

Human ADSC xenograft through IL-6 secretion activates M2 macrophages responsible for the repair of damaged muscle tissue.

BACKGROUND: Adipose-derived mesenchymal stromal cells (ADSCs) are multipotent stromal cells. The cells secrete a number of cytokines and growth factors and show immunoregulatory and proangiogenic properties. Their properties may be used to repair damaged tissues. The aim of our work is to explain the muscle damage repair mechanism with the utilization of the human adipose-derived mesenchymal stromal cells (hADSCs). METHODS: For the hADSCs isolation, we used the subcutaneous adipose tissue collected during the surgery. The murine hind limb ischemia was used as a model. The unilateral femoral artery ligation was performed on 10-12-week-old male C57BL/6NCrl and NOD SCID mice. The mice received PBS- (controls) or 1 × 106 hADSCs. One, 3, 7, 14 and 21 days after the surgery, we collected the gastrocnemius muscles for the immunohistochemical analysis. The results were analyzed with relevant tests using the Statistica software. RESULTS: The retention time of hADSCs in the limb lasted about 14 days. In the mice receiving hADSCs, the improvement in the functionality of the damaged limb occurred faster than in the control mice. More new blood vessels were formed in the limbs of the mice receiving hADSCs than in limbs of the control mice. hADSCs also increased the infiltration of the macrophages with the M2 phenotype (7-AAD-/CD45+/F4/80+/CD206+) into the ischemic limbs. hADSCs introduced into the limb of mice secreted interleukin-6. This cytokine stimulates the emergence of the proangiogenic M2 macrophages, involved, among others, in the repair of a damaged tissue. Both macrophage depletion and IL-6 blockage suppressed the therapeutic effect of hADSCs. In the mice treated with hADSCs and liposomes with clodronate (macrophages depletion), the number of capillaries formed was lower than in the mice treated with hADSCs alone. Administration of hADSCs to the mice that received siltuximab (human IL-6 blocker) did not cause an influx of the M2 macrophages, and the number of capillaries formed was at the level of the control group, as in contrast to the mice that received only the hADSCs. CONCLUSIONS: The proposed mechanism for the repair of the damaged muscle using hADSCs is based on the activity of IL-6. In our opinion, the cytokine, secreted by the hADSCs, stimulates the M2 macrophages responsible for repairing damaged muscle and forming new blood vessels.

Nuclear magnetic resonance spectroscopy reveals metabolic changes in living cardiomyocytes after low doses of ionizing radiation.

Several lines of evidence indicate that exposure of heart to ionizing radiation increases the risk of cardiotoxicity manifested by heart dysfunction and cardiovascular diseases. It was initially believed that the heart is an organ relatively resistant to radiation. Currently, however, it is suspected that even low doses of radiation (< 2 Gy) may have a negative impact on the cardiovascular system. Cardiotoxicity of ionizing radiation is associated with metabolic changes observed in cardiac cells injured by radiation. In this study, we used human cardiomyocytes as a model system, and studied their metabolic response to radiation using high-resolution magic angle spinning nuclear magnetic resonance techniques (HR-MAS NMR). Human cardiomyocytes cultured in vitro were exposed to ionizing radiation and their survival was assessed by clonogenic assay. Changes in apoptosis intensity and cell cycle distribution after the irradiation were measured as well. NMR spectra of cardiomyocytes were acquired using Bruker Avance 400 MHz spectrometer at a spinning rate of 3200 Hz. Survival of cardiomyocytes after NMR experiments was assessed by the Trypan blue exclusion assay. Exposure of cardiomyocytes to small doses of ionizing radiation had no effect on cell proliferation potential and intensity of cell death. However, analysis of metabolic profiles revealed changes in lipids, threonine, glycine, glycerophosphocholine, choline, valine, isoleucine, glutamate, reduced glutathione and taurine metabolism. The results of this study showed that ionizing radiation affects metabolic profiles of cardiomyocytes even at low doses, which potentially have no effect on cell viability.