Developing treatments that target cancer metabolic reprogramming is gaining momentum, but it is often neglected that these approaches are not specific only to proliferating cancer cells. Metabolic therapies inevitably exert a systemic effect, influencing also the non-neoplastic cells, including tumor microenvironment (TME), which in turn may completely redirect the final outcome of the disease. Indeed, emerging literature warns that cancer cell metabolism may have crucial consequences on the phenotype of different non-malignant cell types within the tumor [1
]. For example, tumor cells may engage in metabolic strategies such as nutrient competition to avoid immune cytotoxicity [2
]. On the other hand, targeting cancer metabolic reprogramming may trigger compensatory responses leading to TME-related resistance mechanisms [3
]. Thus, strategies directed against cancer metabolism need to be tested in models that allow a proper assessment of the TME effect on tumor progression.
The bone microenvironment has recently been recognized as essential in determining the fate of osteosarcoma development [4
], even to the extent that targeting TME has been suggested as an efficient strategy to fight the disease [6
]. In this context, it is interesting to note that studies evaluating the efficacy of antimetabolic therapies in osteosarcoma, such as those against mitochondrial complex I (CI), suggest the TME may play a role in defining the response to treatment [3
]. In line with these findings, we have recently reported that targeting CI arrests progression of osteosarcomas, converting them into low-proliferative, oncocytoma-like lesions, and demonstrated that the loss of hypoxia inducible factor 1-alpha (HIF-1α) is accountable for the antitumorigenic effects of CI dysfunction [3
]. However, the latter was associated with an abundance of tumor associated macrophages (TAMs), whose depletion improved the anti-cancer efficacy of metformin, a known CI inhibitor [3
]. Thus, even though targeting CI is being recognized as a valid anti-cancer strategy associated with various antitumorigenic effects [10
], at the same time CI inhibitors seem to elicit conflicting consequences on TME which alter the therapy response [16
In murine xenografts, TME is usually taken into account by an orthotopic implant, which consents cancer cell proliferation within the native environment. For some tumor types, this approach is relatively simple, as in the case of injecting breast cancer cells in the mouse mammary fat-pad [17
], or in hepatocellular carcinoma setting, where orthotopic tumor models involve a minor surgery [18
]. The establishment of in situ osteosarcomas is particularly challenging, as the surgical procedures to reach the tibia or femur are invasive, and the injection is difficult due to bone stiffness, requiring drilling bone plateau and potentially resulting in the leakage of cancer cells [19
]. Engineering approaches today may be used to create ectopic grafts resembling bone tissue environment [5
], allowing not only tumor progression in the native tissue, but also generation of human TME, which is important in the setting where human cancer cells are being investigated [21
]. Such approaches are becoming crucial for appropriate investigation of osteosarcoma development, since the bone tumor fate was shown to be influenced by the inoculation environment [5
Here we take advantage of new methods to humanize and modulate ectopic osteosarcoma graft, with the aim to understand whether CI-deficiency induces changes in bone specific non-neoplastic cells during cancer progression. This pseudo-orthotopic approach established that abundance of cancer associated fibroblasts (CAFs) and TAMs is a hallmark of CI-deficient TME, and identified a peculiar association between targeting CI and osteocyte/osteoblast preservation.
2. Materials and Methods
2.1. Cell Lines
Osteosarcoma 143B Tk− cells were purchased (#CRL-8303, ATCC, LGS Standards, Milan, Italy) and cultured at low passages (<50) in in Dulbecco’s modified Eagle medium (DMEM) High Glucose (#ECM0749L, Euroclone, Milan, Italy), supplemented with 10% FBS (#ECS0180L, Euroclone), L-glutamine (2 mM, #ECB3000D, Euroclone), penicillin/streptomycin (1x, #ECB3001D, Euroclone) and uridine (50 µg/mL, #U3003, Sigma-Aldrich, Milan, Italy), in an incubator with a humidified atmosphere at 5% CO2 and 37 °C. The cell origin was authenticated using AMPFISTRIdentifiler kit (#4322288, Applied Biosystems, Monza, Italy) and their STR profile corresponded to their putative background. Genome editing for generation of NDUFS3 knock-out was performed using zinc finger endonucleases purchased from Sigma-Aldrich (#CKOZFND15168, Milan, Italy), according to the manufacturer’s instructions.
Primary human mesenchymal stroma cells (hMSCs) were purchased (#PT-2501, Lonza, Slough, UK) and grown in alpha Minimun Essential Medium (αMEM) (#32571-028, Gibco, Paisley, UK), hMSC-specific FBS (10%) (#12662-029, Gibco, Paisley, UK), penicillin/streptomycin (1x, #ECB3001D, Euroclone), and used at low passages (<5).
2.2. Establishing Pseudo-Orthotopic Osteosarcomas
Most steps were performed as previously described [22
]. All pre-surgical procedures were performed in sterile conditions. Gelfoam gelatin sponges (2 cm × 6 cm × 7 mm) (Pfizer, Kalamazoo, MI, USA) were sectioned into 24 pieces, washed with ethanol 70%, rehydrated in sterile PBS and placed in a 24-well plate. hMSC cells (5 × 105
in 50 µl) were injected with a syringe (29G) and left to attach for 4 hours in a 37 °C incubator. Culture media was added and the cells were left to grow for 7 days. On day 8 osteosarcoma cells (105
in 30 µl) were injected into the scaffolds and left to attach before adding fresh culture media. On day 9 the scaffolds were clothed following previously described protocol [22
]. Each scaffold was allocated in a 15 mL tube and 8 μL of bone morphogenic protein 2 (BMP-2) (Noricum, Tres Cantos, Spain) (reconstituted in acetic acid 50 mM at 5 μg/μL) were added. Then, 30 μL of thrombin from human plasma (Sigma, Dorset, UK) (reconstituted in 2% CaCl2
at 20 U/mL) and 20 μL of fibrinogen from human plasma (Sigma) (PBS reconstituted at 4 mg/100 mL) were incorporated. Solidification was allowed during 30 min in cell culture conditions before proceeding with in vivo implantation.
Surgery was performed in aseptic conditions. Five to six-week old female Rag1−/- FVB/n mice available at The Francis Crick Institute Biological Research Facility (London, UK) were used. The animals were treated according to institutional guidelines and regulations and experiments performed in accordance with UK Home Office regulations under project license PPL number P83B37B3C. A bilateral implantation was performed. In detail, 2 hours before surgical procedure caprofren (Rimadyl, Zoetis, Leatherhead, UK) anti-inflammatory and pain-killer drug was administrated to each animal, both subcutaneously and in the drinking water. Anesthesia was induced with 2.5% isoflurane and O2 at 2–4%. A wide section of fur from the back was shaved. Then skin was sterilized twice with surgiscrub. For each scaffold implantation, 0.5 cm vertical incision was made 1 cm away from the spine on each side of the animal. With forceps, a pocket under the skin was made in the incision, down the side of the animal. A scaffold was inserted, making sure it was placed deep within the pocket, and then incisions were dried and glued (3M surgical glue, Vetbond, St Paul, MN, USA). Buprenorphine (Vetergesic, Alstoe, York, UK) post-operative analgesia was administrated subcutaneously. Animals were placed in a pre-warmed cage and left to recover. After surgery, animals were checked frequently for their well-being. Rimadyl in the drinking water was removed 48 hours after surgery. Mice were sacrificed either at 30 or at 60 days post implantation.
2.3. Micro Computed Tomography Imaging
Samples were scanned using a SkyScan-1176 μCT scanner (Bruker MicroCT, Kontich, Belgium). The X-ray source was operated at 40 kV and 600 μA, no filter was used. The scans were made over a trajectory of 180° with a 0.5° step size with a 8.57μm pixel size. The images were reconstructed using nRecon (Bruker MicroCT, Kontich, Belgium) and further analysed using CTan (Bruker MicroCT).
Tumor tissue was processed following standard immunohistochemistry protocols. Before embedding, the samples were decalcified with 17% EDTA (Osteosoft, #101728, Merck Millipore, Watford, UK) for 7 days. Hematoxylin/eosin coloration was performed following standard protocol and collagen fibers staining with the Masson’s Trichrome Stain Kit (#25088, Polysciences, Hirschberg an der Bergstrasse, Germany). The following primary antibodies were used: mouse monoclonal anti-HIF-1α (1:100, #610959, BD Biosciences, Berkshire, UK); mouse monoclonal anti-KI-67 (1:100, #M7240, Dako, Agilent, Cernusco sul Naviglio, Italy); rat anti-endomucin (1:200, #SC-65495, Santa Cruz, DBA, Segrate, Italy); mouse anti-SMA (1:750, #M0851, Dako) and rat monoclonal F4/80 (1:100, #14-4801, eBiosciences, ThermoFisher, Life Technologies, Monza, Italy). For evaluation of KI-67 positive nuclei, only cancer cells were counted at 60× magnification in one hot spot area per tumor, avoiding stromal infiltrations and necrotic tissue. Macrophages (F4/80+) were counted at of 20× magnification in three fields of view (FOV) per tumor. The macrophages located close to trabecular bone were counted by considering F4/80 positive cells touching the bone matrix. The macrophages infiltrating the tumor tissue were counted by avoiding tumor front, trabecular bone and necrotic tissue. Osteocytes and osteoblasts were counted in three consecutive FOV at 60× magnification, in proximity to the trabecular bone, starting from the hot spot area.
Immunofluorescent staining included 15 min citrate antigen retrieval (10 mM sodium citrate, pH = 6) at 95 °C, 10 min blocking with goat serum (#156046, Abcam, Cambridge, UK) at RT, 1 hour incubation with primary antibodies at RT (rat anti-endomucin (1:200, #SC-65495, Santa Cruz) and mouse anti-SMA (1:750, #M0851, Dako), 40 min incubation with Alexa Fluor (ThermoFisher, Life Technologies, Monza, Italy) secondary antibodies at RT (488-goat anti-mouse diluted 1:500 and 555-goat anti-rat diluted 1:350) and mounting with Vectashield Antifade Mounting Medium containing DAPI (#H-1200, Vector Laboratories, Peterborough, UK). Vessel size was evaluated by measuring the longer diameter of 20 endomucin positive cells per tumor and avoiding areas of collective fibroblast infiltration. Immature vessels (Endo+SMA−) were counted in five FOV at 20× magnification per tumor.
2.5. Flow Cytometry
Xenograft samples (approximately 50 mm3) were digested immediately after the sacrifice for 40 minutes at 37 °C with Liberase TL (#5401020001, Sigma), Liberase TM (#5401135001, Sigma) and DNaseI (#DN25, Sigma) in HBSS and passed through a 100 µm strainer. Hypotonic lysis with Red Blood Cell Lysis Buffer (#11814389001, Sigma) was performed and remaining cells were washed with MACS buffer (2 mM EDTA, 0.5% BSA in PBS), blocked using FcR Blocking Reagent (#130-092-575, Miltenyi, Surrey, UK) and incubated with panels of pre-labelled antibodies. In parallel, spleen, lung and a control tumor tissue were digested together and stained for Fluorescence Minus One (FMO) reading which was considered while setting the gating strategy. The following panels were used: Panel 1 (for analysis of the tumor macrophage, neutrophil and dendritic cell contribution): anti anti-CD45-APC (clone 30-F11, #17-0451-82, eBioscience), anti-CD11b-ef450 (clone M1/70, #48-0112-82, eBioscience), anti-F4/80-FITC (clone BM8, #123108, BioLegend, London, UK), anti-Ly6G-APC780 (clone RB6-8C5, #47-5931-80, eBioscience), anti-CD11c-PE (clone n418, #12-0114-81, eBioscience); Panel 3 (for analysis of M1/M2 protumorigenic macrophages): anti-CD45-APC780 (clone 30-F11, #47-0451-80, eBioscience), anti-F4/80-ef450 (clone BM8, #48-4801-82, eBioscience), anti-CD206-APC (clone C068C2, #141707, BioLegend). All antibodies were used at 1:100 dilution, apart from the anti-CD45 which was diluted 1:300. Between 300,000-500,000 cells were stained. Dead cells were stained with DAPI and gated out for analyses. Absolute cell abundance was defined as their percentage among all live cells (%Live). Relative cell abundance was defined as their percentage among populations indicated in the figure panels. The samples were run on LSRFortessa cell analyzer (BD Biosciences) and data was analyzed by BD FACSDIVA Software (BD Bioscience) and Flow Jo (Tree Star Inc., Ashland, OR, USA) software.
2.6. Cytokine Profiling
Xenograft-derived cell cultures were generated by a 10-day cultivation of liberase-digested tissue in basal conditions. Supernatant (0.5 mL) was taken 2 days after medium renewal from a 500,000 cells cultured and analyzed with human Proteome Profiler Array kit (ARY005B, R&D Systems, Abingdon, UK) following manufacturer’s instructions. ImageJ was used for quantification of the dot blots.
2.7. Statistical Analyses
GraphPad Prism version 7 (GraphPad Software Inc., San Diego, CA, USA) was used to perform statistical tests and create bar plots and graphs. Unless stated otherwise, a two-tailed unpaired Student’s t-tests assuming equal variance were performed to compare averages. For each experiment, p-values (* p < 0.05, ** p < 0.01, *** p < 0.001) are indicated in the graphs.
In this study, we show that by using a humanized niche model of the osteosarcoma graft, an additional level of information about the tumor histology is achieved with respect to canonical subcutaneous implant. The peculiar bone microenvironment preservation in CI-deficient tumors highlights that parenchymal cells are an important component of TME, warning they should not be neglected when investigating cancer progression. Indeed, functionally relevant cancer associated parenchyma has recently been described also in the setting of breast cancer metastases [27
The differences regarding bone specific cell types appreciated depending on the condition tested, allow to hypothesize that osteocytes and osteoblasts may influence the response to therapies designed against CI. Targeting CI in osteosarcomas grown in the humanized bone reduced tumorigenic potential of 143B cells, albeit not as strikingly as observed in our previously described experimental settings [3
]. Among other, these milder consequences may be due to the osteocyte/osteoclast-specific functions. Their preservation was particularly associated with the later stages of tumor progression, at which the antitumorigenic effect of targeting CI was less appreciated, suggesting that bone-specific non-neoplastic cells may be involved in promoting osteosarcoma survival. The possible mechanisms of growth support may be relative to essential metabolites exchange between cancer and TME cells, as previously suggested [28
]. On the other hand, TME may sustain cancer cell proliferation by promoting angiogenesis. In this context, CI deficiency was also associated with the abundance of TAMs, that have been called into play to provide angiogenic factors when cancer cell-autonomous HIF1 signals are absent [25
]. Indeed, the observation of TAM abundance and vasculature typical of myeloid cell proangiogenic activity in the context of the orthotopic CI-deficient xenografts corroborates our previous findings [3
], suggesting that targeting CI in 143B osteosarcoma prevents HIF1-MIF activation, leading to TAM accumulation and vascular architecture remodeling.
Further investigation is required to understand the significance of these data, by using larger animal cohorts and CI inhibitors, rather than the genetic disruption of the complex, since a drug will inevitably act on TME cell populations as well [16
]. Moreover, adaptive immunity should be taken into consideration by using immunocompetent models. In this context, it is important to note that a murine bone niche could be easily generated by populating the scaffold with murine MSCs. Interestingly, a study evaluating the effects of CI inhibitor metformin in immunocompetent settings reported reduced number of myeloid derived suppressor cells and TAMs in osteosarcomas [9
]. The authors worked with intra-dermal grafts, therefore it would be of interest to understand what is the effect of CI inhibition on bone specific TME cells, such as osteocytes and osteoclasts. This would be particularly important since, in the bone tumor context, the fate of cancer cells in vivo depends on the type of graft that is being used as a model. For example, subcutaneous injection of transformed bone marrow mesenchymal cells is associated to development of leiomysarcoma-like tumors, while the intrabone transplantation of the same cells induced metastatic osteoblastic osteosarcoma, underlining the importance of signals elicited by the bone TME [5
Taken together, preservation of osteocytes and osteoblasts observed upon targeting CI points to the importance of setting up appropriate tumor models, which take into consideration the origin of the cancer in question, since apart from immune cell populations, the parenchymal cells of the TME may also influence neoplastic development.