Caroline E Reynaud
email: caroline.reynaud@inserm.fr

Laura Ferreras

Paola Di Mauro

Casina Kan

Martine Croset

Edith Bonnelye

Floriane Pez

Clémence Thomas

Géraldine Aimond

Antoine E Karnoub

Marie Brevet

Philippe Clézardin
email: philippe.clezardin@inserm.fr

Lysyl oxidase is a strong determinant of tumour cell colonization in bone

Keywords: metastasis, IL-6, osteoclast, osteoblast

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The lysyl oxidase (LOX) family of secreted copper-dependent amine oxidases consists of five paralogs: LOX and LOX-like 1-4 (LOXL 1-4). The primary function of the LOX family is to catalyse the covalent crosslinking of collagen and elastin in the extracellular matrix, thereby increasing insoluble matrix deposition and tensile strength [START_REF] Barker | The rationale for targeting the LOX family in cancer[END_REF]. Increased expression of LOX and LOXL2 has been consistently reported in various cancer types (colorectal, breast, prostate, lung, bladder) [START_REF] Barker | The rationale for targeting the LOX family in cancer[END_REF][START_REF] Pez | The HIF-1inducible lysyl oxidase activates HIF-1 via the Akt pathway in a positive regulation loop and synergizes with HIF-1 in promoting tumor cell growth[END_REF][START_REF] Barker | LOXL2-mediated matrix remodeling in metastasis and mammary gland involution[END_REF][START_REF] Baker | Lysyl oxidase enzymatic function increases stiffness to drive colorectal cancer progression through FAK[END_REF][START_REF] Cox | LOX-mediated collagen crosslinking is responsible for fibrosis-enhanced metastasis[END_REF]. LOX and LOXL2 are closely associated with desmoplastic areas at the invasive front of infiltrating tumours [START_REF] Barker | The rationale for targeting the LOX family in cancer[END_REF]. They mediate collagen/elastin crosslinking that increases extracellular matrix stiffness, a process which is associated with enhanced integrin-mediated mechanotransduction coupled to increased tumour cell invasion in breast and colorectal cancers [START_REF] Barker | The rationale for targeting the LOX family in cancer[END_REF][START_REF] Barker | LOXL2-mediated matrix remodeling in metastasis and mammary gland involution[END_REF][START_REF] Levental | Matrix crosslinking forces tumor progression by enhancing integrin signaling[END_REF]. LOX-and LOXL2-mediated collagen crosslinking are also responsible for enhancing outgrowth in metastatic xenograft models of breast cancer [START_REF] Cox | LOX-mediated collagen crosslinking is responsible for fibrosis-enhanced metastasis[END_REF][START_REF] Barry-Hamilton | Allosteric inhibition of lysyl oxidase-like-2 impedes the development of a pathologic microenvironment[END_REF]. Additionally, LOX enhances the metastatic trait of breast tumour xenografts in animals by stimulating TWIST1 transcription in tumour cells [START_REF] El-Haibi | Critical role for lysyl oxidase in mesenchymal stem cell-driven breast cancer malignancy[END_REF]. In the same vein, hypoxia-inducible factor-1 (HIF-1) induces the expression of several members of the LOX family in breast cancer cells, including LOX and LOXL2, which then catalyses collagen crosslinking in the lungs, facilitating the recruitment of bone marrow-derived cells and the subsequent colonization of the pulmonary tissue by tumour cells [START_REF] Cox | LOX-mediated collagen crosslinking is responsible for fibrosis-enhanced metastasis[END_REF][START_REF] Erler | Lysyl oxidase is essential for hypoxia-induced metastasis[END_REF][START_REF] Erler | Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche[END_REF][START_REF] Wong | Hypoxia-inducible factor 1 is a master regulator of breast cancer metastatic niche formation[END_REF]. Furthermore, we have shown that LOX synergises with HIF-1 in promoting in vivo growth of colorectal tumours [START_REF] Pez | The HIF-1inducible lysyl oxidase activates HIF-1 via the Akt pathway in a positive regulation loop and synergizes with HIF-1 in promoting tumor cell growth[END_REF]. These experimental results [START_REF] Pez | The HIF-1inducible lysyl oxidase activates HIF-1 via the Akt pathway in a positive regulation loop and synergizes with HIF-1 in promoting tumor cell growth[END_REF][START_REF] Barker | LOXL2-mediated matrix remodeling in metastasis and mammary gland involution[END_REF][START_REF] Cox | LOX-mediated collagen crosslinking is responsible for fibrosis-enhanced metastasis[END_REF][START_REF] Levental | Matrix crosslinking forces tumor progression by enhancing integrin signaling[END_REF][START_REF] Barry-Hamilton | Allosteric inhibition of lysyl oxidase-like-2 impedes the development of a pathologic microenvironment[END_REF][START_REF] El-Haibi | Critical role for lysyl oxidase in mesenchymal stem cell-driven breast cancer malignancy[END_REF][START_REF] Erler | Lysyl oxidase is essential for hypoxia-induced metastasis[END_REF][START_REF] Erler | Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche[END_REF][START_REF] Wong | Hypoxia-inducible factor 1 is a master regulator of breast cancer metastatic niche formation[END_REF] probably explain why increased expression of LOX and LOXL2 in primary tumours (colorectal, breast, lung, prostate) is associated with distant relapse and poor survival [START_REF] Barker | The rationale for targeting the LOX family in cancer[END_REF]. Indeed, targeting of LOX and LOXL2 with the LOX inhibitor β-aminoproprionitrile (βAPN) or a function-blocking antibody (AB0023), respectively, is efficacious at reducing metastatic tumour burden in xenograft models of cancer [START_REF] Barry-Hamilton | Allosteric inhibition of lysyl oxidase-like-2 impedes the development of a pathologic microenvironment[END_REF][START_REF] Bondareva | The lysyl oxidase inhibitor b-aminopropionitrile diminishes the metastatic colonization potential of circulating breast cancer cells[END_REF]. Thus, there is a body of experimental evidence indicating that LOX and LOXL2 facilitate the development of metastases in distant organs, such as the lungs, liver, and brain. Surprisingly, little is known regarding the role of LOX proteins during bone metastasis formation.

In bone metastasis, metastatic cancer cells residing in the bone marrow alter the functions of bone-resorbing (osteoclasts) and bone-forming (osteoblasts) cells and hijack signals coming from the bone matrix [START_REF] Weilbaecher | Cancer to bone: a fatal attraction[END_REF]. By disrupting the physiological balance between bone resorption and bone formation, metastatic cells therefore promote skeletal destruction. The detection rate of disseminated tumour cells (DTC) in the bone marrow from colorectal cancer (CRC) patients receiving curatively intended surgery is 27% and their presence is associated with a poor clinical outcome [START_REF] Pantel | Differential expression of proliferation-associated molecules in individual micrometastatic carcinoma cells[END_REF][START_REF] Flatmark | Disseminated tumour cells as a prognostic biomarker in colorectal cancer[END_REF]. This detection rate is relatively high and comparable to what is observed in bone-tropic cancers such as breast cancer (about 30%) [START_REF] Pantel | Differential expression of proliferation-associated molecules in individual micrometastatic carcinoma cells[END_REF], which is in intriguing contrast to the low incidence of overt bone metastases in CRC [START_REF] Qiu | Pattern of distant metastases in colorectal cancer: a SEER based study[END_REF]. A current hypothesis gaining ground is that DTCs may be representative of dormant tumour cells with the ability to escape dormancy upon receiving appropriate stimuli from the microenvironment [START_REF] Sosa | Mechanisms of disseminated cancer cell dormancy: an awakening field[END_REF].

Given the role of LOX in driving collagen crosslinking and extracellular matrix stiffness [START_REF] Barker | The rationale for targeting the LOX family in cancer[END_REF], we hypothesized that LOX in CRC may be one of the factors that endow DTCs with full competence for aggressive colonization in bone. Interestingly, it has been very recently shown during the course of this study that hypoxia-induced LOX in breast cancer cells generates premetastatic osteolytic lesions in animals through the stimulation of NFATc1, a master regulator of osteoclastogenesis [START_REF] Cox | The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase[END_REF]. Here, we investigated the role of LOX and its regulator HIF-1α in CRC bone metastases.

Materials and Methods

Reagents, human cancer cell lines, and animals. All chemicals were obtained from Sigma-Aldrich, unless otherwise specified. Primer pairs used for quantitative real-time PCR analysis are listed in Supplementary Table S1. Human Hct116, HT29 and LS174Tr CRC cell lines (ATCC) were tested before their usage for authentication by DNA fingerprinting using short tandem repeat (STR) method in 2010. The mutation in the ras proto-oncogene was also tested in 2014. Hct116 cells were maintained in RPMI-1640-Glutamax (Invitrogen) with 10% (v/v) FBS, 10mM HEPES, 1mM sodium pyruvate, and 0.1mM non essential amino acids (Invitrogen) in humidified atmosphere at 37 °C. HT29 and LS174Tr cells were grown in DMEM-glutamax (Invitrogen), 10% (v/v) FBS and 50μg/ml gentamycin. These cells were first transduced with lentiviral shRNA particles targeting LOX (shLOX) or a control sequence (shCTL) then co-transduced with particles containing an empty vector (EV) or a LOX-expressing vector (LOX+) under the control of a doxycycline (dox)-inducible promoter, as previously described [START_REF] Pez | The HIF-1inducible lysyl oxidase activates HIF-1 via the Akt pathway in a positive regulation loop and synergizes with HIF-1 in promoting tumor cell growth[END_REF]. Transduced cell lines initially called shCTL/EV, shLOX/EV (cells silenced for endogenous LOX) and shCTL/LOX+ (cells overexpressing LOX in an inducible manner) (2) were named, respectively, Ctrl, LOX-and LOX+ for brevity. Additionally, Ctrl and LOX+ Hct116 cells were co-transduced with lentiviral shRNA particles targeting HIF-1a (Ctrl/HIF1α-and LOX+/HIF1α-, respectively), as previously described [START_REF] Pez | The HIF-1inducible lysyl oxidase activates HIF-1 via the Akt pathway in a positive regulation loop and synergizes with HIF-1 in promoting tumor cell growth[END_REF].

Four-week-old female Balb/c immunocompromised mice were purchased from Janvier (Saint-Berthevin, France). Animals were maintained in a 12-h light-dark cycle and given free access to food and water. All procedures involving animals, including their housing and care, the method by which they were culled, and experimental protocols were conducted in accordance with a code of practice established by the local ethical committee of the University of Lyon.

Immunohistochemistry. CRC tissue specimens (primary tumors and bone metastases)

were obtained from the Department of Pathology, Hospices Civils de Lyon. Tumour sections (5µm) were de-paraffinized, treated with pepsin for 15-min at 37°C (Zymed, Invitrogen® Carlsbad USA), and processed for immunohistochemical staining using a LOX antibody (1:250 dilution; ab31238 Abcam).

LOX activity measurement. LOX activity was measured using a fluorescent assay, according to manufacturer's instructions (ab112139 Abcam).

ELISA. Conditionned media from LOX-and LOX+#Hct116 were collected, and IL-6 was measured by ELISA, according to manufacturer's instructions (Clinisciences).

Protein extraction and Western blotting. Proteins were extracted in RIPA buffer (Sigma), electrophoresed on a SDS-polyacrylamide gel (Life technologies), then transferred onto nitrocellulose membranes (Millipore, Billerica, MA) and proteins were probed with a primary antibody against STAT3 (HP1001671, 1:300 dilution, Sigma), phosphoSTAT3 (9145, 1:1,000 dilution, Cell Signaling), Src (clone GD11, 1:1,000 dilution, Millipore), Src Y416 (Clone 916, 1:1,000 dilution, Millipore), FAK (05-537, 1:1,000 dilution, Upstate), phosphoFAK (44-624, 1:1,000 dilution, Biosource), β-actin (A5060, 1:15,000 dilution, Sigma) or tubulin (T5168, 1:2,000 dilution, Sigma). After incubation with primary antibodies, membranes were incubated with horseradish peroxide (HRP)conjugated secondary goat anti-mouse (1:2,000 dilution, BioRad) or goat anti-rabbit (1:2,500 dilution, BioRad) antibody. Immunostaining was performed with enhanced chemiluminescence (ECL) detection system (Perkin Elmer).

Functional cell-based assays. Ctrl, LOX-and LOX+ Hct116 cells (4x10 6 /ml), treated or not treated with 350μM βAPN or 100 μg/ml tocilizumab, were cultured for 48h in complete RPMI medium without phenol red. Conditioned media were then collected, centrifuged and stored as aliquots at -80°C until used. LOX production was analyzed by western blotting, as previously described [START_REF] Pez | The HIF-1inducible lysyl oxidase activates HIF-1 via the Akt pathway in a positive regulation loop and synergizes with HIF-1 in promoting tumor cell growth[END_REF].

For cell adhesion assays, experiments were conducted as previously described [START_REF] Noblesse | Lysyl oxidase-like and lysyl oxidase are present in the dermis and epidermis of a skin equivalent and in human skin and are associated to elastic fibers[END_REF]. Briefly, 96-well tissue culture plates were coated with increasing concentrations of type-I collagen or fibronectin (0.01mg/cm 2 to 100mg/cm 2 ) and incubated overnight at 4°C. Cells (4×10 4 cells/0.1 ml/well) were allowed to adhere for 20 min at 37°C in a 5% CO2 atmosphere. After washing to remove non-adherent cells, adherent cells were fixed and stained with 0.1% (w/v) crystal violet. The dye was eluted with 2% (w/v) SDS and the optical density quantified at 595 nm. Alternatively, 96-well tissue culture plates were coated with type-I collagen (15 μg/cm 2 ) overnight at 4°C. Following incubation with 1% BSA for 30 min, cells (1×10 5 cells/0.1 ml/well) were incubated for 1h at 37°C in a 5% CO2 atmosphere. After washing, adherent cells were fixed and stained with hematoxylin and fuschin.

For tumour spheroid-formation assays, 1x10 4 /0.3 ml Hct116 cells (Ctrl, LOX-and LOX+) were seeded in CytoCapture Chambers, Big Hexagonal Cavities (diameter 250µm) (PAA). Cells were grown in suspension in 0.3mL of MammoCult TM basal medium containing 10% (v/v) MammoCult TM proliferation supplement (Stem Cell), 0.0004% (w/v) heparin (Stem Cell), 1µg/mL hydrocortisone, 1mM glutamine, 50U/mL penicillin, 50µg/mL streptomycin (Life Technologies), with or without 350µM βAPN. Tumour spheroids were imaged under a microscope (Confocal-Leica SP5 X) and quantified using Fiji software. Spheroids of at least 60µm in diameter were counted.

Osteoclastogenesis assay. Experiments were conducted as described previously [START_REF] Croset | TWIST1 expression in breast cancer cells facilitates bone metastasis formation[END_REF].

Briefly, bone marrow cells from 6-week-old OF1 mice were cultured for 7 days in α-MEM medium (Invitrogen) supplemented with 10% (v/v) fetal calf serum (Invitrogen), 20 ng/ml M-CSF (R&D Systems) and 200 ng/ml RANKL, alone or in combination (from day 1 to day 7) with the conditioned media from transduced Hct116 cells (Ctrl, LOX+ and LOX-) (25 μg/ml) or with recombinant LOX (150 ng/ml). After 7 days in culture, mature osteoclasts were enumerated under a microscope on the basis of the number of nuclei (more than three nuclei) and TRAP activity.

NFATc1 staining. Cultured osteoclasts on coverslips were fixed for 15 min in 4% PFA then permeabilized with 0.1% Triton X-100 in PBS for 10 min. Saturation was performed with 5% normal goat serum in 0.1% PBS-Tween20 (NGS) for 2h. Monoclonal antibody to NFATc1 (SC-7294, Santa Cruz), diluted 1:100 in NGS, was incubated 1h at room temperature and, after washing, coverslips were further incubated for another 1h with a secondary goat anti-mouse antibody coupled to Alexa Fluor 488 (1:300 dilution in PBS). Coverslips were then mounted in FluorSave Reagent (Calbiochem).

Osteoblastogenesis assay. Experiments were conducted as previously described [START_REF] Fradet | A new murine model of osteoblastic/osteolytic lesions from human androgen-resistant prostate cancer[END_REF].

Briefly, calvaria of 3-day-old OF-1 mice were dissected then cells were enzymatically isolated by sequential digestion with collagenase and plated into 24-well plates. After 24-h incubation, α-MEM medium containing 10% fetal bovine serum (Life technologies, Carlsbad, CA, USA) was changed and supplemented with 50μg/ml ascorbic acid (Sigma-Aldrich, Buchs, Switzerland) and with or without conditioned medium from transduced Hct116 cells (Ctrl, LOX+ and LOX-). Medium was changed every other day for 21 days. 10mM sodium β-glycerophosphate (Sigma-Aldrich, Buchs, Switzerland) was added for the last week of the experiments. At day 21, bone mineralized nodules were fixed and stained with von Kossa staining.

Animal studies. CRC cell lines (5x10 5 cells in 100 μl PBS) were inoculated intraarterially into anesthetised female Balb/c nude mice. When specified, 300 μl of conditioned medium from transduced Hct116 cells were intraperitoneally injected daily into mice. For LOX inhibition, animals were treated with 0.2% (w/v) βAPN, supplied in the drinking water. For blockade of IL-6 receptor, animals were treated every other day with tocilizumab (50 mg/kg, i.p.). The progression of skeletal tumour burden was monitored by whole-body bioluminescence imaging (NightOwl, Berthold Technologies), following subcutaneous administration of luciferin (100 mg/kg in PBS; Promega) 10 min prior to imaging. The progression of osteolytic lesions in the skeleton of anesthetised animals was monitored by radiography, using a cabinet X-ray system (MX-20; Faxitron X-ray Corporation). The area of osteolytic lesions was measured using Explora-Nova Morpho Expert software. Animals were sacrificed on day 7 or 35 after tumour cell inoculation, and hind limbs were collected for histology and histomorphometric analyses.

Bone histology and histomorphometry. Bone histology and histomorphometric analysis of bone tissue sections were performed on decalcified, 5-μm bone-tissue sections stained with Goldner's Trichrome, as previously described [START_REF] Croset | TWIST1 expression in breast cancer cells facilitates bone metastasis formation[END_REF].

Histomorphometric measurements [bone volume (BV)/tissue volume (TV) and tumour volume (TuV)/soft tissue volume (STV) ratios] were performed in a standard zone of the tibial metaphysis, situated at 0.5 mm from the growth plate, including cortical and trabecular bone. The BV/TV ratio represents the percentage of bone tissue. The TB/STV ratio represents the percentage of tumour tissue. Additionally, osteoclasts within bone tissue sections were stained using a TRAP activity kit assay (Sigma). The resorption surface (Oc.S/BS) was calculated as the ratio of TRAP-positive trabecular bone surface (Oc.S) to the total bone surface (BS) using the image analysis system MorphoExpert (Explora-nova).

Ex vivo micrometastasis experiments. Ex-vivo micrometastasis experiments were conducted as previously described [START_REF] Croset | TWIST1 expression in breast cancer cells facilitates bone metastasis formation[END_REF]. Animals were culled on day 7 or day 35 after tumor cell inoculation. Hind limbs were collected and tibiae and femurs were minced then soaked in an enzyme cocktail containing 300U/ml type-I collagenase and 100 U/ml hyaluronidase (StemCell Technologies) in DMEM medium for 2 hours at 37°C. After incubation, bone marrow cell suspensions were seeded in 6-well plates and cultured in complete medium. After 1-day, cultured cells were placed under puromycin selection for 2 weeks, enabling the selective outgrowth of antibiotic-resistant tumour cells. Tumour cell colonies were then fixed, stained with 0.5% (v/v) crystal violet and counted.

Clinical correlation analyses. Gene expression data and clinical annotations were

downloaded from The Cancer Genome Atlas for colorectal cancer and previously published datasets downloaded from the Gene Expression Omnibus (GSE16125, GSE33113, GSE41258, GSE17536, GSE31595, GSE12945) (see Supplementary Table S2).

Statistical Analysis. All experimental data are presented as mean values ± SD.

Statistical comparisons of values were made using the Mann-Whitney U-test. Diseasefree survival Kaplan Meier analyses were performed using the log-rank (Mantel-Cox) test. Correlation analyses were performed by the Spearman Rank test and the Pearson correlation. All tests were two-sided, and P values less than 0.05 were considered statistically significant.

Results

LOX expression in colorectal cancer is clinically associated with poor prognosis.

As a first step towards evaluating the role of LOX and its regulator HIF-1α in CRC bone metastases, we conducted a meta-analysis in a cohort of CRC patients (n = 552) and found that high LOX expression in primary tumours was associated with poor overall survival (p = 0.0109) and poor relapse-free survival (p = 0.02) (Supplementary Figs. S1a andS1b). Additionally, high LOX expression was a negative determinant of relapse-free survival, irrespective of HIF-1α levels (Supplementary Fig. S1c). Using immunohistochemistry with a anti-LOX antibody, we examined 5 CRC patients with bone metastases, including 3 patients for whom we had pairs of primary tumours and their matching bone metastases. Although all of the matching primary and metastatic tumours expressed LOX, there was a preferrential moderate-to-strong staining for LOX associated with the carcinoma cells in bone metastases for all 5 patients (Fig. 1 and Supplementary Fig. S1d).

Tumour-secreted LOX in colorectal cancer generates osteolytic lesions in animals.

We next investigated the role for LOX and HIF-1α in CRC bone metastasis formation, using human Hct116 cells (Ctrl) previously transduced for HIF-1α silencing (Ctrl/HIF-1α-) and/or LOX overexpression (LOX+ ; LOX+/HIF-1α-) (2). Compared to Ctrl and Ctrl/HIF-1α-tumour-bearing animals, bioluminescent LOX+ Hct116 cells were readily detected in the hind limbs of animals at day 31-post injection, irrespective of HIF-1 expression (Fig. 2a). Additionally, there was a similar effect on osteolytic lesions in LOX+ and LOX+/HIF-1α-tumour-bearing animals and the extent of lytic lesions was 2.5-fold higher than that of animals bearing Ctrl-or Ctrl/HIF-1α-tumors (Fig. 2b).

Hence, these data indicated that tumour-secreted LOX promotes osteolytic lesion formation in vivo, irrespective of HIF-1α expression.

To further address the role of LOX in osteolytic lesion formation, we used two additional previously published human CRC cell lines (HT29 and LS174Tr) in which LOX expression has been overexpressed or silenced [START_REF] Pez | The HIF-1inducible lysyl oxidase activates HIF-1 via the Akt pathway in a positive regulation loop and synergizes with HIF-1 in promoting tumor cell growth[END_REF]. The modulation of LOX expression did not affect the expression of other members of the LOX-like family (Supplementary Table S3). We observed that LOX-overexpressing Hc116, HT29 and LS174Tr cells caused osteolytic lesions in animals at day 35-post injection (Fig. 2c). The extent of osteolytic lesions in LOX+ tumour-bearing animals was 3-to 5-fold higher than that of animals bearing Ctrl or LOX-tumours (Fig. 2c). Additionally, the treatment of LOX+ Hct116 tumour-bearing animals with the LOX inhibitor βAPN for 35 days, prolonged bone metastasis-free survival to a level similar to that observed with animals bearing LOX-or Ctrl-Hc116 tumours (Supplementary Fig. S2). Histomorphometric analysis of hind limbs with metastases from animals bearing Hct116 tumours showed that the BV/TV ratio (a measure of the bone volume) was decreased in LOX+ tumour-bearing animals, compared with LOX-and Ctrl tumour-bearing animals (Fig. 2d and Table 1). This difference was accompanied with a substantial increase in the TRAP staining of bone tissue sections of metastatic legs from LOX+ tumour-bearing animals (indicating a stimulation of active-osteoclast resorption surfaces) (Fig. 2d and Table 1). Additionally, there was a dramatic increase in the TuV/STV ratio (a measure of the skeletal tumour burden) in LOX+ tumour-bearing animals, compared with animals bearing Ctrl-and LOX-tumours (Fig. 2d and Table 1).

To further examine LOX-dependency, nude mice were treated with the conditioned medium from LOX+ or LOX-Hct116 cells beginning 1 day (D-1) before intra-arterial inoculation of parental Hct116 cells (D0). The daily treatment with the conditioned medium then continued until day 35 (D35), at which time anesthetised animals were analysed by radiography (Fig. 3a). Radiographs of animals showed that mice treated with the LOX+ conditioned medium had a 6-fold increase in the extent of osteolytic lesions, compared to that observed with the LOX-medium (Figure 3b). The bone marrow of 3 metastatic mice per group was then collected and placed in culture under antibiotic selection, enabling the selective outgrowth of antibiotic-resistant tumour cells (Fig. 3c). The average number of colonies recovered from the bone marrow of mice treated with the LOX+ conditioned medium was 2.5-fold higher than that recovered from animals treated with the LOX-conditioned medium (344 ± 60 vs 121 ± 25 colonies/well) (P = 0.08) (Fig. 3c). Hence, LOX plays a prominent role in supporting osteolytic lesion formation in vivo and tumour cell dissemination ex vivo.

LOX disrupts the balance between bone resorption and bone formation.

By disrupting the physiological balance between bone resorption and bone formation, tumour cells promote skeletal destruction [START_REF] Weilbaecher | Cancer to bone: a fatal attraction[END_REF]. To directly test whether LOX expression in Hct116 cells could influence osteoclast differentiation, we treated primary mouse bone marrow cell cultures with RANKL and M-CSF together with the conditioned medium from Ctrl, LOX-and LOX+ Hct116 cells. Consistent with in vivo data (Fig. 2d), the conditioned medium from LOX+ Hct116 cells stimulated the formation of TRAP-positive multinucleated osteoclasts, compared to conditioned media from Ctrl-and LOX-Hct116 cells (Fig. 4a). As aforementioned, βAPN treatment of LOX+ tumour-bearing animals inhibited osteolytic lesion formation (Supplementary Fig. S2), suggesting that LOX-mediated bone destruction critically depends on LOX's enzymatic activity. In agreement with this suggestion βAPN almost completely inhibited LOX enzymatic activity in the conditioned medium from LOX+ Hct116 cells, when compared to that observed with the conditioned medium from LOX-Hct116 cells (Supplementary Fig. S3). Additionally, βAPN treatment blocked the stimulatory effect of LOX+ on osteoclastogenesis (Fig. 4a), indicating that LOX-induced osteoclastogenesis critically depends on LOX's enzymatic activity.

LOX overexpression induced a robust production of IL-6 by Hct116 cells, whose production was almost totally inhibited by βAPN treatment of LOX+ Hct116 cells (Fig. 4b). Tumour-derived IL-6 can act as an autocrine agent and activate the signal transducer and activator of transcription 3 (STAT3) signalling pathway in CRC cells [START_REF] Hsu | Anti-interleukin-6 receptor antibody inhibits the progression in human colon carcinoma cells[END_REF][START_REF] Lin | STAT3 is necessary for proliferation and survival in colon cancer-initiating cells[END_REF]. In agreement with these findings [START_REF] Hsu | Anti-interleukin-6 receptor antibody inhibits the progression in human colon carcinoma cells[END_REF][START_REF] Lin | STAT3 is necessary for proliferation and survival in colon cancer-initiating cells[END_REF], STAT3 phosphorylation levels were substantially increased in LOX+ Hct116 cells, compared to Ctrl-and LOX-Hct116 cells (Fig. 4c). Additionally, blockade of the IL-6 receptor by tocilizumab abrogated STAT3 phosphorylation in LOX+ Hct116 cells, compared with untreated cells (Fig. 4c). By contrast, the FAK (focal adhesion kinase)/Src tyrosine kinase activities in Hct116 cells were the same whether these cells expressed or did not express LOX (Supplementary Fig. S4). Thus, our data revealed a previously unreported functional link between LOX and IL-6, in which LOX-driven IL-6 production by CRC cells promotes IL-6R/STAT3 signalling pathway. This link extended to the clinic, because LOX and IL-6 significantly correlated across 7 publically available databases on CRC, when analysed independently or in combination (Fig. 4d and Supplementary Fig. S5). The addition of IL-6 to the conditioned medium of LOX-Hct116 cells stimulated osteoclast formation to an extent similar to that observed with the conditioned medium from LOX+ Hct116 cells (Fig. 4e).

Moreover, tocilizumab inhibited osteoclastogenesis induced by the LOX+ conditioned medium, and an additive inhibitory effect on osteoclast formation was observed when combining tocilizumab and βAPN (Fig. 4f). These results (Fig. 4e-f) suggested that both IL-6 and LOX were effective stimulators of osteoclastogenesis. Indeed, in agreement with previous findings [START_REF] Cox | The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase[END_REF], we observed that recombinant LOX (in the presence of RANKL+M-CSF) stimulated the formation of osteoclasts in vitro, compared to control (RANKL+M-CSF) (Fig. 5a). Additionally, it promoted a greater nuclear localization of NFATc1, the master regulator of osteoclastogenesis, than RANKL+MCSF alone (85 vs 30% of positive nuclear staining; P < 0.01) (Fig. 5b). Hence, tumour-secreted LOX and IL-6 were acting in concert to stimulate the generation of differentiated osteoclasts.

To determine whether LOX expression in Hct116 cells could also influence osteoblast differentiation, we treated primary calvarial mouse osteoblasts with the conditioned medium from Ctrl, LOX+ or LOX-Hct116 cells. We found that the conditioned medium from LOX+ Hct116 cells partially inhibited the formation of bone nodules and their mineralization, compared to conditioned media from Ctrl and LOX-Hct116 cells (Fig. 5c-d).

Collectively, our findings suggested that tumour-derived LOX induces an imbalance between bone resorption and bone formation, which leads to bone destruction in vivo.

LOX primes tumour cells for dissemination to the bone marrow

To determine the role of LOX in the settlement of tumour cells in the bone marrow, animals injected with parental Hct116 cells were treated daily from D-1 to D7 with the conditioned medium from LOX-or LOX+ Hct116 cells, with or without tocilizumab or βAPN treatment. The bone marrow was then collected and placed in culture under antibiotic selection, enabling growth of tumour cells that have disseminated to the bone marrow (Fig. 6a). We recovered colony-forming tumour cells in the bone marrow from 7 out of 10 animals treated with the LOX+ conditioned medium, whereas only 2 out of 8 animals treated with the LOX-conditioned medium had tumour cell colonies in the bone marrow (Fig. 6b). Moreover, the average number of colony-forming tumour cells recovered from the bone marrow of the 7 metastatic mice treated with the LOX+ conditioned medium was 4-fold higher than that recovered from the 2 mice treated with the LOX-conditioned medium (Fig. 6b). The treatment of animals with βAPN completely blocked the stimulatory effects of LOX on incidence (1 out of 6 animals) and number of colony-forming tumour cells in the bone marrow (Fig. 6b). In sharp contrast, tocilizumab did not inhibit LOX stimulatory effect on tumour cell dissemination (fig. 6b).

Thus, these data indicate that LOX (irrespective of IL-6) is crucially important in the settlement of tumour cells in the bone marrow.

LOX promotes tumour cell adhesion and survival

We have previously reported that LOX overexpression in breast cancer cells induces bone metastasis, a phenotype associated with its ability to induce the expression of a major epithelial-to-mesenchymal (EMT)-transcription factor, TWIST1 [START_REF] El-Haibi | Critical role for lysyl oxidase in mesenchymal stem cell-driven breast cancer malignancy[END_REF]. Additionally, we have shown that TWIST1 facilitates breast cancer bone metastasis formation [START_REF] Croset | TWIST1 expression in breast cancer cells facilitates bone metastasis formation[END_REF]. We therefore proceeded to explore whether LOX promoted tumour cell colonization in bone via induction of EMT. Hct116 cells expressed detectable levels of genes encoding for EMT-inducing transcription factors, such as TWIST1 and SNAI1 (Snail) (Supplementary Fig. S6). LOX expression in Hct116 cells did not modify the morphological appearance of these cells and it did not modify TWIST1, SNAI1 or vimentin (VIM) expression (supplementary Fig. S6), indicating that LOX in colon cancer cells does not contribute to bone marrow colonization by promoting an EMT.

Early steps of bone metastasis involve the attachment and survival of DTCs to and in the bone matrix [START_REF] Weilbaecher | Cancer to bone: a fatal attraction[END_REF], two properties we examined using in-vitro assays. Collagens constitute 90% of the total protein content in bone, type-I collagen being the most abundant bone extracellular matrix protein [START_REF] Rossert | Type I collagen: Structure, synthesis and regulation[END_REF]. Cell attachment experiments to type-I collagen were therefore performed using Ctrl, LOX-and LOX+ Hct116 cells. As shown in Fig. 6c, there was a significant gain in the attachment of LOX-overexpressing tumour cells to collagen, compared to Ctrl Hct116 cells. The attachment of LOX-deficient tumour cells was lower than that observed with Ctrl Hct116 cells (Fig. 6c). In contrast, the extent of attachment of Hct116 tumour cell lines to fibronectin was the same, irrespective of LOX expression levels (Fig. 6c). Using Hct116 cells silenced for LOX expression (LOX-) as a control to measure baseline cell attachment to type-I collagen, we conducted cell attachment experiments with LOX+ Hct116 cells, in the presence or absence of βAPN. As shown in Fig. 6c, βAPN abrogated the gain of attachment of LOX+ Hct116 cells to type-I collagen (LOX+ βAPN), compared to that observed with untreated cells (LOX+). Hence, these observations suggested that LOX crosslinking activity is important to mediate CRC cell attachment to type-I collagen.

Once tumour cells seed in the bone marrow, they need to survive in this microenvironment. A relevant in vitro feature of CRC cell survival is their ability to grow as colonospheres [START_REF] Shaheen | Spheroid-Formation (Colonosphere) Assay for in Vitro Assessment and Expansion of Stem Cells in Colon Cancer[END_REF]. We thus asked whether Ctrl, LOX-, and LOX+ Hct116 cells could form colonospheres in serum-free medium. Indeed, LOX+ Hct116 cells formed a higher number of colonospheres than the two other Hct116 cell lines, and growth of LOX+ Hct116 cells as colonospheres was significantly inhibited in the presence of βAPN (Supplementary Fig. 7 and data not shown). Thus, LOX can provide CRC cells with a survival advantage in the bone marrow.

Discussion

A large body of preclinical and clinical evidence has shown that LOX expression in tumours facilitates the progression of several cancers and the development of metastases to distant organs, such as the lungs, liver, and brain [START_REF] Barker | The rationale for targeting the LOX family in cancer[END_REF][START_REF] Pez | The HIF-1inducible lysyl oxidase activates HIF-1 via the Akt pathway in a positive regulation loop and synergizes with HIF-1 in promoting tumor cell growth[END_REF][START_REF] Baker | Lysyl oxidase enzymatic function increases stiffness to drive colorectal cancer progression through FAK[END_REF][START_REF] Barry-Hamilton | Allosteric inhibition of lysyl oxidase-like-2 impedes the development of a pathologic microenvironment[END_REF][START_REF] Erler | Lysyl oxidase is essential for hypoxia-induced metastasis[END_REF][START_REF] Erler | Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche[END_REF]. Indeed, LOX has been identified as an important regulator of hypoxia-induced tumour progression via a HIF-1α-dependent mechanism in numerous cancer types [START_REF] Barker | The rationale for targeting the LOX family in cancer[END_REF][START_REF] Erler | Lysyl oxidase is essential for hypoxia-induced metastasis[END_REF][START_REF] Erler | Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche[END_REF][START_REF] Wong | Hypoxia-inducible factor 1 is a master regulator of breast cancer metastatic niche formation[END_REF].

Surprisingly, aside from a very recent report that hypoxia-induced LOX promotes bone metastasis of breast cancer [START_REF] Cox | The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase[END_REF], there was no evidence as to whether this is a generalised function; i.e. if tumour-derived LOX participates in the development of bone metastasis in other cancer models, such as colon cancer. We have previously shown that LOX synergizes with HIF-1α in promoting in vivo growth of colorectal cancers [START_REF] Pez | The HIF-1inducible lysyl oxidase activates HIF-1 via the Akt pathway in a positive regulation loop and synergizes with HIF-1 in promoting tumor cell growth[END_REF]. In the present study, we found that LOX was expressed by tumour cells in the bone marrow from CRC patients with bone metastases and that, irrespective of HIF-1 expression, tumour-secreted LOX promoted settlement of CRC cells in the bone marrow and induced a robust expression of IL-6 by these tumour cells. In turn, both LOX and IL-6 played a prominent role in enhancing differentiation of mature osteoclasts. In breast cancer, it has been shown that LOX stimulates the generation of differentiated osteoclasts through the activation of NFATc1 [START_REF] Cox | The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase[END_REF]. LOX secreted from CRC cells also promoted the nuclear translocation of NFATc1 in osteoclasts. Additionally, LOX inhibited osteoblast differentiation, thereby creating an imbalance between bone resorption and bone formation. Based on these findings we propose a model in which LOX supports CRC cell dissemination in the bone marrow and LOX-driven IL-6 production by CRC cells impairs bone homeostasis, thereby promoting osteolytic lesion formation in vivo (Fig. 6d).

Having shown that tumour-secreted LOX drives osteolytic lesion formation in vivo, we then attempted to determine the specific steps of bone colonization to which LOX functions are manifested. It has been previously reported that LOX enhances the metastatic trait of breast tumours in animals by stimulating TWIST1 expression in tumour cells [START_REF] El-Haibi | Critical role for lysyl oxidase in mesenchymal stem cell-driven breast cancer malignancy[END_REF]. Additionally, LOX overexpression in breast cancer has been associated with EMT of tumour cells [START_REF] Han | Potential options for managing LOX+ ER-breast cancer patients[END_REF]. Here, LOX overexpression in Hct116 cells did not modify the morphological appearance of these cells and it did not modify the expression of transcription factors TWIST1 and SNAI1, indicating that LOX in CRC cells did not contribute to tumour cell dissemination in bone by promoting an EMT. Instead, our results established that LOX directly plays a stimulatory role in the survival of CRC cells in the bone marrow by enhancing their attachment to type-I collagen. These results are in agreement with previous findings [START_REF] Barker | The rationale for targeting the LOX family in cancer[END_REF][START_REF] Oskarsson | Metastatic stem cells : sources, niches, and vital pathways[END_REF][START_REF] Laklai | Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression[END_REF], showing that LOX is a major enhancer of extracellular matrix stiffness and that, during metastasis initiation, tumour cells respond to tensile forces imposed by extracellular matrix stiffness with activation of AKT, FAK, Src and/or STAT3 signalling pathways. We previously showed that LOX induces AKT phosphorylation in Hct116 cells [START_REF] Pez | The HIF-1inducible lysyl oxidase activates HIF-1 via the Akt pathway in a positive regulation loop and synergizes with HIF-1 in promoting tumor cell growth[END_REF]. Here, FAK and Src activation in CRC cells occurred irrespective of LOX expression, whereas STAT3 was activated by LOX through a IL-6/IL-6R-dependent signalling pathway. Additionally, blockade of the IL-6 receptor by tocilizumab abrogated STAT3 phosphorylation in LOX+ Hct116 cells. However, tocilizumab did not inhibit tumour cell dissemination in the bone marrow in vivo, whereas the LOX inhibitor βAPN did. Interestingly, extracellular matrix tension can directly drive STAT3 activation in pancreatic ductal carcinoma cells [START_REF] Laklai | Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression[END_REF]. It is conceivable that LOX promotes STAT3 activation in CRC cells by increasing extracellular matrix stiffness. Our findings do not preclude the possibility that other members of the LOX family could contribute to the dissemination of CRC cells in the bone marrow.

However, endogenous mRNA levels of LOXL1, LOXL2, LOXL3 and LOXL4 in Hct116 cells were only barely detectable (2), and LOX overexpression in these tumour cells did not modify expression levels of other LOX family members (Supplementary Table S3).

Although overt bone metastasis is rare in colon cancer and frequent in breast cancer, the detection rate of DTCs in the bone marrow from CRC patients is comparable to what is observed in breast cancer [START_REF] Pantel | Differential expression of proliferation-associated molecules in individual micrometastatic carcinoma cells[END_REF]. It seems therefore that colon cancer DTCs should harbor a mechanism to grow out, but this appears to be somehow blocked in colon cancer patients. Only 5 CRC patients with bone metastases were studied here.

However, all of them had LOX-positive tumour cells in the bone marrow. Additionally, LOX overexpression endowed CRC cells with full competence for aggressive colonization in bone in vivo. Environments that are rich in type-I collagen may be critical for DTCs, promoting their transition from dormancy to metastatic growth [START_REF] Barkan | Metastatic growth from dormant cells induced by a col-I-enriched fibrotic environment[END_REF]. Given the role of LOX in driving collagen crosslinking [START_REF] Barker | The rationale for targeting the LOX family in cancer[END_REF], it is conceivable that LOX participates to metastatic outgrowth of DTCs.

In conclusion, our findings collectively show that LOX supports CRC cell dissemination in the bone marrow and they reveal a novel mechanism through which LOX-driven IL-6 production by CRC cells impairs bone homeostasis. We believe this is a crucially important observation, which supports targeting LOX for metastasis prevention. 1 BV/TV = bone volume-to-tissue volume ratio; TuV/STV = tumour volume-to-total soft tissue volume ratio;

Osteoclastogenesis Osteolysis

Type-I Collagen

Tumour cell adhesion

Bone metastasis formation

OC.S/BS = active osteoclast-resorption surface per trabecular bone surface.; TRAP = tartrate-resistant acid phosphatase.

Figure 1 .

 1 Figure 1. LOX expression in pairs of human primary colorectal carcinoma and their matching bone metastasis. Immunohistochemistry was performed using 3 pairs of patients' tumour specimens. A strong immunostaining for LOX was observed in neoplastic cells (arrows) and, to a less extent, in the desmoplastic tumor stroma. In bone (*), immunostaining for LOX was observed in osteocytic lacunae. Scale bar: 1-mm.

Figure 2 .

 2 Figure 2. High LOX expression in colorectal cancer cells promotes osteolytic lesion formation in vivo. (a) Whole body bioluminescence imaging of animals at day 31 after intra-arterial inoculation of luciferase-expressing Hct116 cells. Ctrl: parental Hct116 cells. LOX+: LOX-overexpressing Hct116 cells. Ctrl/HIF-1α-: shRNA-HIF1α-expressing

Figure 3 .

 3 Figure 3. Systemic delivery of tumour-secreted LOX enhances osteolytic lesion formation and tumour cell outgrowth in the bone marrow. (a) Schematic representation of the experimental protocol. Parental Hc116 cells were inoculated intraarterially to 4-week-old female Balb/c nude mice. Animals received a 5-week daily treatment with the conditioned medium from LOX-or LOX+ Hct116 cells, starting one day before tumour cell inoculation. Tumour-bearing mice were analyzed by radiography

Figure 4 .

 4 Figure 4. LOX-driven IL-6 production by colorectal cancer cells stimulates osteoclastogenesis. (a) In vitro osteoclast differentiation of murine bone marrow cells treated with M-CSF+RANKL in combination with the conditioned medium from Ctrl-, LOX-or LOX+ Hct116 cells. The conditioned medium from LOX+ Hct116 cells treated with βAPN was also tested. Mature osteoclasts were quantified as multinucleated (more than 3 nuclei), TRAP-positive cells. (b) ELISA measurement of IL-6 in the conditioned medium from Ctrl-, LOX-, LOX+ or LOX+/βAPN Hct116 cells. (c) Immunoblot analysis of phosphorylated STAT3 in Ctrl-, LOX-, and LOX+ Hc116 cells, treated or not treated with tocilizumab. Graphs show intensities of phosphorylated STAT3 relative to total STAT3. Tubulin was used as a control for equal loading. (d) Correlation analysis of LOX and IL-6 expression intensities from 7 databases on colon cancer (n = 1,146 patients). (e) Effect of exogenous IL-6 (10 ng/ml) on osteoclastogenesis induced with M-CSF+RANKL in combination with the conditioned medium of LOX-Hct116 cells, compared with that of LOX+ Hct116 cells. (f) Effects of the anti-IL6 receptor antibody tocilizumab (100 ng/ml) and of β-APN (350μM) on osteoclastogenesis induced with M-CSF+RANKL in

Figure 5 .

 5 Figure 5. LOX stimulates osteoclastogenesis and inhibits osteoblastogenesis. (a) Recombinant LOX (rLOX; 150 ng/ml) enhanced osteoclast differentiation induced by RANKL+M-CSF. (b) Conditioned medium from LOX+ Hc116 cells promoted the nuclear translocation of NFATc1 in osteoclasts, compared to conditioned medium from parental Hct116 cells (Ctrl). Nuclei are stained blue with DAPI. (c) and (d) In vitro differentiation of primary mouse calvaria cells cultured in osteogenic conditions with conditioned media from Ctrl, LOX-and LOX+ Hct116 cells. Bone mineralized nodules (stained black with von Kossa staining) were counted under light microscopy. Data are mean ± SD. *,**: P < 0.05 and 0.01, respectively.

Figure 6 .Fig. 2 :

 62 Figure 6. Systemic delivery of tumour-secreted LOX promotes the settlement of colorectal cancer cells in the bone marrow. (a) Schematic representation of the experimental protocol. Parental Hc116 cells were inoculated intra-arterially to nude mice, then animals were treated with conditioned media of LOX-or LOX+ Hct116 cells (treated or not treated with βAPN or tocilizumab). (b) Graphs showing for each group the average number of tumour cell colonies recovered from the bone marrow. Each bar corresponds to a single mouse. Data are mean ± SD. **: P < 0.01. (c) Attachement of Ctrl-, LOX-and LOX+ Hct116 cells to increasing concentrations of adhesive substrates (collagen, fibronectin). Data are mean ± SD. *,**: P < 0.05 and 0.01, respectively. (d) Schematic representation of the role of LOX during bone metastasis formation. Tumoursecreted LOX enhanced tumour cell attachment to type-I collagen and induced IL-6 production which, in turn, acted as an autocrine agent and activated IL-6R/STAT3

Fig. 3 :Fig. 5 :Fig. 6 :

 356 Fig. 3: Reynaud et al.

Table 1 .

 1 Effects of LOX overexpression or silencing in Hct116 colorectal cancer cells on bone metastasis formation in animals.Values are mean ± SD. Values of p (two sided) are for pairwise comparison with the control group (Ctrl) using the Mann-Whitney U test.

	Cell		Histomorphometry		TRAP staining
	line
		BV/TV (%)	p	TuV/STV (%)	p	OC.S/BS (%) p
	Ctrl	26.5 ± 2.3 (n=8)	-	9.8 ± 6.4 (n=8)	-	31 ± 4.1 (n=5) -
	LOX+ 13.9 ± 2.2 (n=8)	0.0006	57.6 ± 5.5 (n=8)	0.0016	73 ± 6 (n=7)	0.0017
	LOX-	28.3 ± 2.9 (n=7)	0.53	2.6 ± 2.1 (n=7)	0.33	19 ± 8.3 (n=3) 0.09

Acknowledgements

We acknowledge Dr Cyril Confavreux (Lyon, France) for providing tocilizumab and Dr. Sabine Riethdorf (Hamburg, Germany) for conducting immunofluorescence experiments on CRC cells. We also thank Dr Klaus Pantel (Hamburg, Germany) and Dr Catherine Panabières (Montpellier, France) for valuable discussion.

CR acknowledges the support of the Ligue Contre le Cancer (Comité du Rhône) and CNRS. PC is supported by INSERM, the University of Lyon, the Grand Prix de la Recherche Ruban Rose and INCa (grant n°: 2014-164). CK is a recipient of a Marie Curie individual fellowship from the Horizon 2020 European Programme under agreement number n°655777. Additionally, this work was supported by the LabEX DEVweCAN (ANR-10-LABX-61) of Université de Lyon, within the program "Investissements d'Avenir" (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR).


Covadonga Lucas-Torres

Hélène Roumes

Véronique Bouchaud

Anne-Karine Bouzier- Sore

Alan Wong
email: alan.wong@cea.fr

Metabolic NMR mapping with microgram tissue biopsy

Keywords: metabolic mapping, HR-µMAS, NMR, glioma, biopsy. Abbreviations

This study explores the potential of profiling µg-scaled soft tissue biopsy by NMR spectroscopy. The important elements of high resolution and high sensitivity for the spectral data are achieved through a unique probe, HR-µMAS, which allowed a comprehensive profiling to be performed on µg-tissue for the first time under MAS conditions. Thorough spatially resolved metabolic maps were acquired across a coronal brain slice of rat C6 gliomas, which rendered the delineation of the tumor lesion. The results present a unique ex vivo NMR possibility to analyze tissue pathology that cannot be fully explored by the conventional approach, HR-MAS and in vivo MRS. Aside from the capability of analyzing a small localized region for tracking its specific metabolism, it could also offer the possibility to carry out longitudinal investigations on live animals due to the feasibility of minimally invasive tissue excision.

Introduction

MRI has been a vital imaging modality for tumor diagnosis and prognosis due to its amenity of revealing the spatial contrast of the tissue in a non-invasive manner. However, it often relies on molecular spectroscopic technique, in vivo MRS, to unravel the underlying metabolic profiles and pathways for facilitating the characterization of the tumor subtypes and grades. [START_REF] Horská | Imaging of brain tumors: MR spectroscopy and metabolic imaging[END_REF][START_REF] Mandal | Brain metabolic mapping with MRS: A potent noninvasive tool for clinical diagnosis of brain disorders[END_REF] Multivoxel MRS (MRS imaging) is a useful strategy in tissue pathology for providing the spatial metabolic coverage [START_REF] Wu | Metabolomic imaging for human prostate cancer detection[END_REF][START_REF] Taylor | Metabolic Imaging in Humans[END_REF] to understand cancer metabolic reprogramming. However, the spectral interpretation is generally strained by the insufficient resolution due to the acquisition under a static sample condition. Consequently, it commonly resorts to ex vivo HR-MAS NMR to support the interpretation of the in vivo spectra. [START_REF] Opstad | Correlations between in vivo 1 H MRS and ex vivo 1 H HRMAS metabolite measurements in adult human gliomas[END_REF][START_REF] Kaebisch | Applications of high-resolution magic angle spinning MRS in biomedical studies I-cell line and animal models[END_REF][START_REF] Tugnoli | A comparison between in vivo and ex vivo HR-MAS 1H MR spectra of a pediatric posterior fossa lesion[END_REF][START_REF] Tugnoli | Ex vivo HR-MAS MRS of human meningiomas: a comparison with in vivo 1H MR spectra[END_REF] Despite the good spectral resolution by HR-MAS, there have been only few attempts of spatially resolved studies (multi-voxel or -sampling) in cancer research. Notably, Leo et al.

performed the first HR-MAS analysis of different regions in a human glioblastoma. [START_REF] Cheng | Quantification of microheterogeneity in glioblastoma multiforme with ex vivo high-resolution magic-angle spinning (HRMAS) proton magnetic resonance spectroscopy[END_REF] Recently, this team carried out a comprehensive metabolic mapping on a resected human prostate tumor. [START_REF] Dinges | Metabolomic prostate cancer fields in HRMAS-MRS-profiled histologically benign tissue vary with cancer status and distance from cancer[END_REF] In another study, Righi et al. acquired a linear spatial metabolic profile across an excised rat glioma-bearing slice. [START_REF] Righi | Spatially resolved bioenergetics and genetic reprogramming through the brain of rats bearing implanted C6 gliomas as detected by multinuclear high-resolution magic angle spinning and genomic analysis[END_REF] However, one limitation with HR-MAS is the use of an extensive sampling mass (5-10 mg) for compensating the low sensitivity in NMR spectroscopy, which can hinder the deduction of relevant information in a small spatial region. Recently, a new µMAS probe technology, denoted as HR-µMAS, has been introduced. [START_REF] Lucas-Torres | HR-µMAS NMR-based metabolomics: localized metabolic profiling of a garlic clove with µg tissues[END_REF][START_REF] Lucas-Torres | Current Developments in uMAS NMR analysis for metabolomics[END_REF] It targets solely on µg-scaled soft tissues with the capability of acquiring high resolution spectral data without compromising the sensitivity. Unlike the ultra-fast spinning µMAS probe for solids, the HR-µMAS probe is devised to give minimal effect of the magnetic susceptibility toward the µgtissue, allowing high-resolution acquisition to be achieved under the sample-spinning condition. This study examined the HR-µMAS application to microbiopsies. It detailed the metabolic mapping of rat C6 glioma-bearing slices to facilitate the delineation of the tumor lesion. The C6 glioma model was strategically chosen for the proof of concept because of its homogenous nature and the well-characterized MR profiles, which allowed for an unbiased evaluation of the profiling capability of HR-µMAS, which uses µg-tissue.

Experimental section

Animal treatment and glioma implantation

Six male Wistar rats (160-220 g body weight) were used: two controls (C1 and C2), and four tumor-bearing rats (T1-T4). T1-T4 tumors were produced by stereotaxic injection in the left caudate nucleus (part of the basal ganglia) of C6 glioma cells, derived from N-nitrosomethylurea-induced rat glioblastoma. [START_REF] Bendaz | Differentiated rat glial cell strain in tissue culture[END_REF] Rats were anesthetized with isofluorane (4% induction, 2% during surgery) and secured in the stereotaxic apparatus (Stoeling, Germany).

The scalp was cleaned with Betadine (Sarget, France) and local injection of lidocaine was performed (local anesthesia). The skull was then exposed by a midline scalp incision and a 1mm diameter hole was drilled at a position 3.5-mm left from Bregma. Then, 10 6 glioma cells in 10 µl were injected into the left hemisphere at a depth of 5.5 mm. The craniectomy was sealed with bone wax, and the scalp was sutured. In vivo MRI and MRS were periodically performed to follow the tumor growth. Acquisitions were performed with ParaVision 6.0. MRS spectral data were processed separately using Bruker Topspin 3.6.1.

In vivo

Slice resection

Rats were euthanized using brain-focused microwaves (5 kW, 1 s, Sacron8000, Sairem), which stopped all enzymatic and metabolic activities, to avoid post-mortem metabolism and artifacts such as anaerobic lactate production. [START_REF] Sampol | Glucose and lactate metabolism in the awake and stimulated rat: a 13 C-NMR study[END_REF] Brains were then surgically removed, and a 3-mm coronal slice centered on Bregma was rapidly excised, placed in a biopsy cassette, and dipped in liquid nitrogen. The 3-mm thickness was chosen to accommodate a tissue-filled µMAS rotor for an optimal NMR sensitivity. For T3 and T4, an additional slice (adjacent to that used in HR-µMAS) was excised for histopathology. Table S1 lists a summary of the slices (C1, C2 and T1-T4) and their usages.

Histopathology

Histopathology was carried on the adjacent slice of T3 and T4 using a classical Hematoxylin-Eosin staining protocol. Slices were immediately post-fixed for 24 h (4 °C, in PFA 4%). Fixed slices were then cryoprotected by sucrose immersion (48 h in a 20% sucrose solution, followed by 24 h in a 30% solution, 4 °C) and frozen in liquid N 2 vapor. The 20 μm-thick slices were stained with a hematoxylin solution (5 min), washed in distilled water (10 s), dipped in eosin solution (3 min) and rinsed twice (in distilled water). Sections were finally dehydrated in 95%

(1 min), then in 100% ethanol (2 min) and mounted on coverslips in Depex mounting medium (Sigma Aldrich). Brain sections were observed and images were taken using an optical microscope (Leica STP6000).

ex vivo 1 H HR-µMAS NMR

Sample-preparation. Following the guidelines, [START_REF] Lucas-Torres | General guidelines for sample preparation strategies in HR-µMAS NMR-based metabolomics of microscopic specimens[END_REF] the resected slice was placed inside a biopsy cassette with 1´1 mm slots for imprinting the orderly grid pattern to facilitate the sampling (Figure S1). The sampling was carried out using a microbiopsy punch (World Precision Instruments) on a frozen slice, and immediately injected into the 1-mm rotor with few drops of D 2 O/TSP (internal reference). The entire procedure was carried out on a cold platform (Figure S1) and limited to 15-20 minutes. Samples with a longer preparation were automatically discarded.

NMR acquisition. A sacrificed sample was used for optimization (shimming and pulsesequence adjustments). NMR experiments were carried out on a Bruker Avance II, operating at a 1 H frequency of 499.16 MHz with a custom-built JEOL 1-mm 'HR'-μMAS under a sample spinning between 5000-5500 ± 20 Hz, and with a constant temperature of 20 ± 2 °C. The NMR spectra were acquired with a Carr-Purcell-Meiboom-Gill (cpmgpr1d) pulse sequence (TE 80 ms; water-suppression recycle delay 1 s; spectral width 20 ppm; data point 32 K). Four subsequent spectra were collected for each sample, each with 320 averaging with a 13-minute acquisition period. The spectral processing was performed using Bruker Topspin 3.6.1 with a zero-filling and a line broadening of 0.3 Hz. The 1 H chemical shift was referenced to the alanine doublet at 1.48 ppm. Figure S2 shows a comparison of the resolutions between the conventional HR-MAS approach and HR-µMAS. It also shows no apparent signal degradation among the four subsequent spectra. The summed spectra were used in the multivariate and semiquantification analyses.

Data analysis

Multivariate. Processed spectral data were uploaded into MestreNova v8.1. Spectra were aligned, and the water region (4.15-5.10 ppm) was removed. Each spectrum was normalized to the signal of tCr (3.06 to 3.02 ppm), and divided into sub-ppm (0.01 ppm) buckets from 5.20 to 0.77 ppm. The resultant matrix was submitted to SIMCA P13 and scaled by Pareto's distribution (power-law probability distribution). PCA and OPLS-DA were performed to identify the tumor profile. PLS regression was applied to locate the lesion. Models were validated by assessing the values of R 2 X, R 2 Y, and Q 2 as well as by the p-values obtained from cross-validation (level of confidence <0.05) to avoid overfitting. It should be noted that we applied PLS regression because of the accessibility in SIMCA (other regressions algorithms can offer similar, or better, prediction accuracy, such as a combined PCA and canonical variates analysis (CVA), which has been used on the MRS imaging data for delineating prostate tumors in an excised human tissue 3 ).

Semi-Quantification. Due to the differences in the spin relaxation (T 1 and T 2 ) of metabolic signals, we reported the metabolic ratio Q[metA/metB] = (A metA /A metB )´(N metB /N metA ), where A met and N met are the corresponding signal integral and its number of detected protons, respectively.

The deconvolution was carried out by MestreNova v8.1. A representative fitting is shown in Figure S3, and the quantified ratios are summarized in Tables S2-4.

Results and discussion

HR-µMAS NMR of brain tissue

Figure 1 shows a spectral comparison between ex vivo HR-µMAS NMR and in vivo MRS with a sampling voxel of 1´1´3 mm for HR-µMAS and 2.5´2´2.5 mm for MRS. Despite the fact that acquisitions were carried out at different magnetic fields (7 T for in vivo MRS and 11.7 T for HR-µMAS), the spectral effect of MAS is evident. The FWHM of a singlet at 3 ppm in HR-µMAS is significantly narrower with 0.02 ppm as compared to that of 0.6 ppm in in vivo MRS.

Moreover, with the same acquisition time, the SNR for HR-µMAS is higher than that for in vivo MRS. Taking into account the sampling voxel, HR-µMAS renders a crude 7-fold enhancement in volume-sensitivity SNR (mm 3 •Ömin•ÖB 3 ). In other words, it would require a 7 2 , therefore a 49, longer acquisition time for in vivo MRS to achieve the same SNR level obtained with HR-µMAS with µg-tissue volume. This enhancement is ascribed by the use of a µ-detection solenoid and the resultant spectral resolution achieved by the HR-µMAS probe.

Note that the µMAS for solids would not offer the same level of resolution because of the presence of the substantial magnetic susceptibility that will arise from the probe head.

Metabolic NMR profile of gliomas

PCA and OPLS-DA were carried out to identify a specific glioma metabolic profile by examining relative metabolite intensities between the tumor and control tissues. Owing to the µg-scaled sampling, this study was able to analyze a total of 34 samples from a single control coronal slice (C1) and 42 from two glioma-bearing slices (T1 and T2) (Figure S1). The PCA score plot in Figure 2 exhibits a clear distinction of spectral data between control and tumor slices. The discrimination was also reflected in the OPLS-DA with R 2 X = 0.843 and Q 2 = 0.752, and with a modest predictive accuracy AUC = 85%. Owing to the homogenous nature of the tumor tissue and its distinct metabolic profile, the discrimination was further improved between the data acquired from the tumor lesion alone and the control with R 2 X = 0.986, Q 2 =0.963 and with a near 100% AUC; thus, the corresponding S-line has unambiguously diagnosed the NMR markers for the tumor: elevated signals in tCho, Tau, Ala, Gly, myo-Inositol, Lac and lipids, while a reduction in NAA and Glu signals were observed. These results are in line with the previous HR-MAS findings, 17 thus validating the NMR profiling strategy with HR-µMAS.

Glioma delineation and characterization

To evaluate the possibility of localizing the tumor lesion through the spatial NMR mapping, we used a linear PLS regression, Y output = X input B, [START_REF] Lee | Patial least squares-discriminant analysis (PLS-DA) for classification of high-dimensional (HD) data: a review of contemporary practice strategies and knowledge gaps[END_REF] by exploiting the 100% AUC model (Figure 2c) as the regression coefficient B to delineate the lesion in the T3 and T4 models. This was carried out by mapping the NMR profiles across the coronal slices, in which the acquired spectral data X input were then submitted to the regression. The outputs Y output provide the predictive degree 0-1, where 1 represents the spectral profile closely resembling those of the lesion used in B (T1 and T2), and likewise, 0 corresponds to those of the control (C1). Figure 3 shows the resulting regression maps. Evidently, the test model C2 is solely mapped with the control profiles, whereas the located tumor lesions are apparent in T3 and T4, which are in agreement with the visible tumor mass on MR images. Interestingly, the regression indicates that the lesion profiles delineated in T4 are more similar to those in B (T1, T2) as compared to those in T3.

The study quantified and evaluated several NMR-identifiable metabolites across the coronal slice of C2, T3 and T4. Figure 4 shows that in both T3 and T4 an increase in tCho/NAA, Lac/NAA, Gly/tCr and Ala/tCr, and a decrease in Glu/tCr were observed in the lesion. In general, the elevated tCho/NAA arises both from a decrease in NAA signal and an increase in tCho. This magnetic resonance signature has been well recognized and often used as a diagnostic marker for characterizing tumoral tissue in animal models [START_REF] Kaebisch | Applications of high-resolution magic angle spinning MRS in biomedical studies I-cell line and animal models[END_REF][START_REF] Righi | Spatially resolved bioenergetics and genetic reprogramming through the brain of rats bearing implanted C6 gliomas as detected by multinuclear high-resolution magic angle spinning and genomic analysis[END_REF] and in humans. [START_REF] Horská | Imaging of brain tumors: MR spectroscopy and metabolic imaging[END_REF][START_REF] Opstad | Correlations between in vivo 1 H MRS and ex vivo 1 H HRMAS metabolite measurements in adult human gliomas[END_REF][START_REF] Kaebisch | Applications of high-resolution magic angle spinning MRS in biomedical studies I-cell line and animal models[END_REF][START_REF] Lee | Patial least squares-discriminant analysis (PLS-DA) for classification of high-dimensional (HD) data: a review of contemporary practice strategies and knowledge gaps[END_REF] The reduction in NAA, a neuronal viability marker, is interpreted as the loss or abnormality of the neuronal tissue, whereas the increased level of tCho is associated with membrane synthesis and degradation (such as occurring during necrosis, for example). Among tCho signal ratios, T4 displays a slightly higher PC/tCho level (0.69 ± 0.09, Table 1) at the lesion along with a lower level of GPC/tCho (0.24 ± 0.09) as compared to those in T3 (0.56 ± 0.04 and 0.38 ± 0.06, respectively). In a previous study on choline metabolism using ex vivo HR-MAS NMR, [START_REF] Righi | 1 H HR-MAS and genomic analysis of human tumor biopsies discriminate between high and low grade astrocytomas[END_REF] it has been found a difference of the PC level and GPC in human tissues between high and low grade astrocytomas, which appeared to be reflected by the changes in the expression of genes of the Kennedy pathway.

A recent HR-MAS study 11 has found that the tumor lesion is metabolically dominated by anaerobic glycolysis (Warburg effect), which is reflected by the production of lactate from pyruvate through lactate dehydrogenase activity. The study has also observed a decrease in glutamate, presumably reflecting neuronal death, since glutamate is the major neurotransmitter in glutamatergic neurons. [START_REF] Rothman | In vivo NMR studies of the glutamate neurotransmitter flux and neuroenergetics: implications for brain function[END_REF] These results (an increase in lactate and a decrease in Glu) are also evident in Figure 4. An increasing level of Glu/tCr from the peritumor to the contralateral region was apparent in both T3 and T4. Interestingly, the growth was more pronounced in T4, where this ratio in the contralateral hemisphere was even higher compared to the one in C2. This alteration has also been observed in the edema surrounding different human gliomas, [START_REF] Ricci | Metabolic findings on 3T 1 H-MR spectroscopy in peritumor brain edema[END_REF] and could signify neuronal death due to a toxic effect of the excess of excitatory neurotransmitter (exitotoxicity). The high content of Lac/NAA found in the T4 lesion suggests a higher grade glioma, which is often accompanied by lactate production that diffuses from the tumor to the peritumoral region. [START_REF] Raschke | Tissue-type mapping of gliomas[END_REF] Glycine (Gly) is an inhibitory neurotransmitter. It has been found to increase in high grade gliomas by in vivo MRS [START_REF] Maudsley | Mapping of glycine distributions in gliomas[END_REF] and ex vivo HR-MAS NMR, [START_REF] Righi | High-resolution magic angle spinning magnetic resonance spectroscopy detects glycine as a biomarker in brain tumors[END_REF] and has been proposed as a tumoral marker. Despite the µg-scaled tissue, the achieved high resolution and sensitivity of HR-µMAS enable to capture the elevated level of Gly/tCr in the lesions.

An elevated level of Ala/tCr in the lesion was also detected, especially in T4. This is consistent with the previous HR-MAS findings in human meningiomas. [START_REF] Aj | Ex-vivo HRMAS of adult brain tumours: metabolite quantification and assignment of tumour biomarkers[END_REF] It was suggested in an early study [START_REF] Van Veelen | Alanine inhibition of pyruvate kinase in gliomas and meningiomas: A diagnostic tool in surgery for gliomas?[END_REF] that Ala inhibits the pyruvate kinase in gliomas and meningiomas, and Ala has been proposed as a potential biomarker for the evaluation of tumor aggressiveness due to its positive presence in glioblastoma multiforme as compared with low grade gliomas. [START_REF] Righi | 1 H HR-MAS and genomic analysis of human tumor biopsies discriminate between high and low grade astrocytomas[END_REF] The tumor grade is determined by the degree of differentiation of the tumor and its cellularity, as well as the presence of cytonuclear atypia, mitotic activity, microvascular proliferation and necrosis. As such, molecular characterization of the gliomas is an important aspect in clinical medicine for making diagnostic and treatment decision based on the grade. In vivo MRS provides metabolic information of the intratumoral region and facilitates distinguishing the glioma grades. The typical markers are tCho, NAA, Cr and lactate, and the lesser common are Gly, myo-Inositol, PC and GPC. For examples, increase in ratios of tCho/NAA, PC/tCho, Gly/tCr and in lipids, and reduction of the GPC/tCho ratio are consistently found in high grade compared to low grade gliomas.

Here, we evaluated the relevant metabolic ratios of the identified lesion in T1-T4. Ratios are summarized in Table 1 and Figure 5, and suggest the following grade ranking T1 » T2 » T4 > T3. The higher grade in T4 compared with T3 is also evident in the histopathology shown in Figure 5b, in which angiogenesis, necrosis and hemorrhages are observed in T4 but not in T3.

Regrettably, histopathology for T1 and T2 were not available to confirm the comparative grades. Interestingly, despite the four glioma-bearing rats underwent the same surgical stereotactic C6-cell implantation, the tumor growth was evidently different; therefore leading to different tumor metabolic patterns among the rats, especially for T3. Similar observations have also been reported. [START_REF] Fonchy | A new gadolinium-based contrast agent for magnetic resonance imaging of brain tumors: Kinetic study on a C6 rat glioma model[END_REF] The previous study -using the same tumor implantation protocolalso observed two distinct patterns of C6 glioma growth; one with capillaries found at the tumor periphery, and the other containing a tumor core in the center, reflecting the different tumor metabolism and necrosis processes.

Concluding Remarks

This work successfully demonstrates the metabolic profiling of µg-scaled soft tissue biopsy under MAS conditions. As a proof of concept, the delineation of the tumor lesion in the gliomabearing rat was carried out by exploiting the new µMAS technology, which permitted highresolution NMR data acquisition of small tissue voxel. These results illustrate a potential toward the acquisition of metabolic information on a small spatial origin; data that can be difficult to obtain from the conventional HR-MAS and in vivo MRS experiments alone. We foresee suitability for examining the spatial patterns of local drug release in the region of interest. Also,

Figure 1 .

 1 Figure 1. Spectral comparison between in vivo MRS (at 7 T) and ex vivo HR-µMAS NMR (at 11.7 T). Spectra correspond to the tumor lesion in T2. Both were acquired with an acquisition time of 13 minutes, but with different sampling voxel, 2.5´2´2.5 mm and 1´1´3 mm, respectively.

Figure 2 .

 2 Figure 2. (a) PCA and (b) OPLS-DA score plots between all sampling data acquired from C1 and T1-T2; (c) OPLS-DA plot between all data in C1 and those in the tumor lesion of T1 and T2; (d) ROC comparison between models in (b) and (c); and (e) OPLS-DA S-line plot of (c). The significant metabolites are annotated with a correlation coefficient p[corr] in parentheses and a Student's t-test p-value (* p < 0.01, ** p < 0.001 and *** p < 0.0001).

Figure 3 .

 3 Figure 3. PLS-regression analyses: (a) A scheme of PLS regression, with the model in Figure 2c as B; (b) in vivo MRI images of the corresponding T3 and T4; (c) Superimposed regression maps with 1´1 sampling pixels. The level of the predicted values is coded with a 32-colour scheme with red being 1, the likeliness to the tumor profile in B, and blue being 0, the likeliness to the control profile.

Figure 4 .

 4 Figure 4. (a) Superimposed metabolite distribution maps across the coronal slice; (b) Linear plots showing the variations of the metabolite ratios across the coronal slice, along the sampling row 5-6 in Figure 3c. The colored regions correspond to the same scheme as in the regression maps Figure 3c.

Figure 5 .

 5 Figure 5. (a) Bar graphs showing the comparison of metabolite ratios found within tumor regions. Values correspond to the arithmetic mean and the error bar to its standard deviation, and are summarized in Table 1. The t-test's p-values are indicated as * p < 0.05 and *** p < 0.0001; (b) Hematoxylin-Eosin histological sections of the adjacent glioma-bearing slices. The corresponding 250µm scale-bar is displayed in the upper images (´2.5) and in lower images (´10).

Figure 1 Figure 2 Figure 3

 123 Figure 1

Figure 4

 4 Figure 4

Figure 5

 5 Figure 5

1 H MRI and MRS

	MRI and MRS were performed on a 7 T Bruker BioSpec scanner (70/20, Ettlingen, Germany)
	using a Bruker 1 H-transmit volume coil. The tumor was visualized using a T 2 -TurboRARE
	sequence (TE 39.96 ms; TR 2500 ms; average 2; repetition 1; echo spacing 13.319 ms; rare
	factor 8; slice thickness 0.8 mm; 20 slices; interspace 0.2 mm; image size 256´256; field-of-
	view 35´35 mm; scan time 2 min 40 s). 1 H MRS was performed using a LASER sequence
	(voxel 2.5´2´2.5 mm; TE 19.26 ms; TR 2.5 s; average 256; acquisition time 12 min).

Table 1 .

 1 A summary of the mean metabolite ratios deduced from the delineated tumor lesion. a

		Lac/NAA tCho/NAA GPC/tCho	PC/tCho	Glu/tCr	GABA/tCr	Gly/tCr	Tau/tCr	Ala/tCr
	T1(n=7)	5.59±2.09 6.61±0.35	0.24±0.14 0.68±0.12 0.67±0.08 0.24±0.02	0.36±0.01 0.78±0.05 0.40±0.01
	T2(n=7)	1.87±1.01 8.17±1.36	0.29±0.08 0.65±0.07 0.82±0.31 0.17±0.12	0.37±0.09 0.90±0.17 0.39±0.11
	T4(n=6)	4.16±1.49 6.30±2.39	0.24±0.09 0.69±0.09 1.19±0.13 0.48±0.12	0.32±0.13 0.87±0.16 0.39±0.07
	T3(n=4)	1.78±0.24 2.26±0.52	0.38±0.06 0.56±0.04 0.70±0.14 0.16±0.06	0.27±0.03 0.75±0.06 0.30±0.0
	C2(n=11) b 0.56±0.21 0.99±0.25	0.31±0.12 0.54±0.10 1.07±0.15 0.31±0.2	0.08±0.02 0.44±0.04 0.12±0.02
	C1(n=13) b 0.75±0.63 0.94±0.27	0.28±0.11 0.55±0.06 0.91±0.20 0.34±0.08	0.10±0.03 0.46±0.09 0.13±0.05
	(a) Test models are highlighted in grey; (b) Taken from the contralateral hemisphere.

Acknowledgments:

We are grateful to JEOL Resonance Inc. for their technical supports on the HR-µMAS probe, and to Prof Luc Pellerin (Univeristé de Poitiers, France) and Dr Yusuke Nishiyama (JEOL, Japan) for reviewing the manuscript. We would also like to thank Dr Florence Fauvelle (GIN, France) for advising about sample preparation.

the notion of µg-scaled tissue excision promotes minimally invasive surgical procedure on animals, which could present a unique possibility of acquiring longitudinal data on live animals.

Like in all analytical technology, limitations are unavoidable. In HR-µMAS, the low detection sensitivity is an intrinsic bottleneck. Despite the fact that µ-solenoid provides an enhancement for µg-tissue analysis, it is still insufficient to analyze low abundant metabolites. To further improve the sensitivity, it needs to be coupled with other enhancement strategy. One would be the cryogenic probe technology, which reduces the thermal noise originating from the electronic circuits. However, the change in the coil susceptibility under the cryo-temperature would greatly complicate the attainment of high resolution data. Another limitation is the sample preparation, since handling a µg-scaled tissue with a tiny rotor is not trivial. A slight deviation could affect the overall data, resulting in non-reproducible data acquisition and consequently would lead to misinterpreted analysis. Therefore, a more robust and automated approach to fill the rotor would enhance its feasibility and reliability, and would facilitate transfer to biopsy research. In spite of these limitations, the remarkable ability of profiling µg-scaled biopsies promotes a new possibility of assessing tissue pathology and could lead to a valuable complementary option to aid data interpretation of in vivo MRS and ex vivo HR-MAS NMR.

Supplementary Materials: Figures include sampling coordinates for HR-µMAS,

representative HR-µMAS spectra and spectral fitting. Summary tables include the resected brain slices, and deduced metabolite ratios in T3, T4 and C2.

Author Contributions: CLT and AW designed and constructed the study, and carried out the NMR measurements and data analyses. AKBS, HR and VB carried out the animal treatments, MRI and data interpretations. CLT and AW drafted the manuscript, and all authors assisted in revisions.

Funding:

The study was supported by Agence Nationale de la Recherche in France, grant number ANR-16-CE11-0023. AKBS also received support from ANR-10-IDEX and ANR-10-LABX-57.

Conflicts of Interest:

The authors declare no conflict of interest.

Data availability:

The data that support the findings of this study are available from the corresponding author upon reasonable request.