Name: Determinazione analitica della funzione mitocondriale degli omogeneizzati tumorali solidi asportati (Video) | JoVE Journal
Uploaded: 2021-08-06T14:30:00.000+00:00
Duration: 11 min 32 s
Description: Scopri un metodo rapido per misurare la funzione mitocondriale nei tumori solidi usando omogeneizzati di tessuto fresco. Questa tecnica semplifica la preparazione campionaria e massimizza la resa mitocondriale per applicazioni cliniche.

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We thank the Pennington Biomedical Research Center Comparative Biology Core staff for animal care. This research was supported in part by the National Institute of Health grants U54GM104940 (JPK) and KL2TR003097 (LAG). All experiments and procedures involving animals were approved by the Pennington Biomedical Research Center Institutional Animal Care and Use Committee.

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<li>Jorgensen, L. B., Overgaard, J., Hunter-Manseau, F., Pichaud, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dramatic+changes+in+mitochondrial+substrate+use+at+critically+high+temperatures%3A+a+comparative+study+using+Drosophila%5BTitle%5D)+AND+%22Journal+of+Experimental+Biology%22%5BJournal%5D)">Dramatic changes in mitochondrial substrate use at critically high temperatures: a comparative study using Drosophila.</a> Journal of Experimental Biology. 224, Pt 6 (2021).</li>
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</ol>

The authors have no conflicts of interest related to this work.

Approaches to evaluating mitochondrial respiration in cancer have largely been limited to in vitro models13,14,15,16. Some success has been achieved in measuring mitochondrial respiration in tumors using chemical permeabilization6,7,17, but there is no uniform, gold-standard approach that can be universally applied and compared across tumor types. Additionally, a lack of consistent data analysis and reporting has limited data generalizability and reproducibility. The method outlined herein provides a simple, relatively quick approach to measure mitochondrial respiration18 in mitochondrial preparations from freshly excised solid tumor specimens. Tumors were grown from orthotopically implanted murine Luminal B, ER&#945;-negative EO771 mammary cancer cells19.
Diligence and care with tissue handling will greatly improve the accuracy and normalization of the oxygen consumption rates. The tissue and mitochondria can be easily damaged if the sample is not kept cold, is not consistently submerged in preservation media, or is overly handled, resulting in suboptimal routine and OXPHOS rates. Additionally, accurate wet weight of the homogenized tissue is of critical importance as this is the primary normalization method. Other normalization methods may be considered, such as total protein or mitochondrial specific markers, such as citrate synthase activity20. Additionally, tissue heterogeneity will need to be addressed, with decisions about tumor regions to include in experiments made a priori. Necrotic, fibrotic, and connective tissue may not homogenize and/or respire well and should be avoided unless intentionally assaying these tumor regions. Notably, the tumor may be very sticky depending on the type and excision region, making accurate weighing and transfer more challenging. The number of strokes used for homogenizations should be optimized to ensure complete preparation of the mitochondria while mitigating damage to the outer mitochondrial membranes.
For improved accuracy and reproducibility, we recommend optimization experiments be performed for the number of strokes for homogenate preparation, tissue concentration, and substrate, uncoupler, inhibitor concentrations. Studies can compare the different number of strokes and how they correspond with response to the addition of cytochrome c within the study as well as the maximal mitochondrial respiratory capacity 21. Although there is a general acceptance that less cytochrome c response is better, as an increase in oxygen consumption after the addition of cytochrome c can indicate damage to the outer mitochondrial membrane, there is no gold-standard as to what this threshold is for every tissue and should be experimentally investigated to ensure the tissue is not being overworked or underprepared. In this tumor tissue, it was found that a cytochrome c response under ~30% did not impair respiratory function. Cytochrome c use becomes critical for accurate quantification of respiratory capacity if the test is positive. In this case, the addition replenishes endogenous cytochrome c, which, if depleted, will cause an underestimation of the respiratory rates.
Tissue concentration titration experiments can be performed over a range of feasible concentrations and, ideally, would be done with SUITs that will be investigated during the study. Respiratory capacity will vary by tumor type and composition. Thus, tumors dense with mitochondria or high respiratory capacity will require lower concentrations (0.5-5 mg/mL). Tumors with few mitochondria or low respiratory capacity will require higher concentrations (7-12 mg/mL). Additionally, SUITs that are long or have highly consumed substrates may need less tissue to prevent reoxygenation of the chamber or ADP limitation. Some tissues will have a linear relationship in oxygen consumption, whereas others will show improved sensitivity and maximal oxidation at certain concentration ranges. The chosen tissue concentration should be optimized to maximize oxygen flux while limiting the number of reoxygenation events. Additionally, it is often better to overestimate the need or aim for the higher end of the concentration range. The inhibitors, which are essential to the quantitation of respiratory fluxes, are more precise when used in larger pools of mitochondria.
Another essential consideration is the concentration of the drugs that are used during the protocols. Changes in homogenate concentration may alter the concentrations of substrates, uncouplers, and inhibitors required for maximal response. Thus, once the optimal concentration range is chosen, an experiment testing the doses required for the SUIT protocol should be performed. Additional ADP can be added to ensure that adenylate concentrations are not limiting to respiratory fluxes. Chemical uncouplers such as FCCP or CCCP will inhibit respiration at higher concentrations22. As such, it is essential to titrate in small amounts to reveal the maximal achieved rate. Inhibitors, such as rotenone and antimycin A, are best used when saturated within the first injection. While optimal concentrations were determined in preliminary experiments, we have also observed treatment-related differences in response to inhibitors and thus often add one additional injection of inhibitors to demonstrate maximal inhibition as the resultant rates serve as the basis for quantification. Chemical inhibition of Ascorbate/TPMD is essential for accurate analytical reduction as TMPD undergoes autooxidation23. We controlled for auto-oxidation of ascorbate/TMPD/cytochrome c through the addition of sodium azide, an established CIV inhibitor. For the Km studies, the addition of rotenone in the presence of succinate alone prevents oxaloacetate accumulation which can inhibit succinate dehydrogenase activity at low concentrations24. The volume and concentration of ADP are highly dependent on the sensitivity of the mitochondria to the prevailing substrate combination. Mitochondrial preparations that are highly sensitive to ADP will require lower starting concentrations. Additionally, validated chemicals and proper drug preparation with attention to pH, sensitivity to light if applicable, and storage temperature are essential for successful experiments.
Instrument setup and routine care are of critical importance for the success of these experiments. Adequate and proper cleaning of the chambers is essential for reproducibility and prevention of biological, protein, inhibitor, or uncoupler contamination. Clark-type electrodes and O2k systems utilize glass reaction chambers which is a significant cost advantage to plate-based systems which rely on consumables. However, the glass chambers must be vigorously cleaned and can be a source of inhibitor contamination in subsequent studies. Incubation with mitochondria-rich specimens during the washing process (isolated heart or liver mitochondria, for example) can reduce the risk of experimental contamination and is recommended in addition to dilution and alcohol-based washing procedures. If consecutive studies are run, cleaning with ethanol and mitochondria minimizes the possibility of inhibitor contamination. Calibration of the oxygen sensor is recommended prior to each experiment to obtain accurate measurements of respiration relative to the prevailing partial pressure of oxygen. If multiple calibrations are not feasible, one calibration a day may be sufficient if the oxygen concentration remains stable and consistent after the washing procedure.
The procedures outlined above leverage the Oroboros O2k instrument for measuring oxygen consumption in tumor tissue within 4 hours of tumor excision using previously designed and optimized preservation solution and respiration media25,26,27. Multiple parameters in this protocol can be modified for subsequent applications. The instrument setup and calibration, the homogenizers used for tissue preparation, and optimal homogenate and chamber oxygen concentration can all be adapted for use on other instruments with oxygen monitoring potential. For example, the chambers were slightly overfilled when adding homogenate, and thus when the chamber is fully closed, the chamber capillary remains full. This will consume&#160;some oxygen in the chamber, but with optimization of sample concentration, we can account for this consumption in determining what oxygen level to start with. Alternatively, the sample can be allowed to equilibrate at ambient oxygen before the chamber is closed, but this will often increase the amount of time before the experiment starts and delay the addition of substrates. While the homogenizers used in this protocol are widely accessible, other commercial homogenization techniques could be employed, such as a tissue shredder or automated homogenizer28.
Additionally, the tissue preparation and instrument procedures can be utilized with a number of different SUITs to study respiratory control by a diversity of coupling and pathway control states29. These SUIT protocols have been developed to measure functional capacity, and thus, the contribution of potential endogenous substrates has no impact on the capacity measurement. We analytically account for non-mitochondrial oxygen consumption and/or residual consumption of the homogenate through subtraction of the antimycin A-rotenone, or sodium azide insensitive rates, as appropriate. Mitochondria can remain viable in BIOPS or similarly constructed preservation solutions for extended periods of time (&#62;24 h) depending on tissue type and intactness30,31. Studies can be carried out in advance to determine the temporal storage limits as OXPHOS of certain substrates may have different limitations. This is essential if the experiment cannot be performed within several hours of tissue excision/biopsy. 37&#176;C is an optimal and physiological temperature for the evaluation of respiratory function in most mammalian systems. However, if the assay temperature appears to interfere with evaluation32, comparative studies may be conducted across a wide temperature range (25-40 &#176;C) to ensure adequate responsiveness. Instrumental constraints may limit the ability to conduct such studies.
Major limitations of the above-described method are 1) the potential for damage to mitochondria through mechanical homogenization, 2) presence of ATPases or other sub-cellular biochemicals in homogenate preparations that can interfere with simultaneous determination of ATP or other variables of interest and may require additional correction methods or inhibitor use33, and 3) evaluation of many samples and/or multiple SUITs per sample is time-consuming as one instrument can accommodate two experiments at a time and requires cleaning and set up in-between successive experiments. Optimization experiments and consistent preparation of samples can minimize substantial mitochondrial damage that would contribute to inconsistent data.
The significance of the method with respect to existing/alternative methods is improved feasibility compared with the amount of starting material, challenge of isolating mitochondria, or technical challenge in permeabilizing tissue. Preparation of homogenates is faster, oxygen is not nearly as limiting, and is less susceptible to variability between personnel compared with permeabilized tissue. Importantly, nearly all sample types are suitable for homogenate preparation which allows for comparative analysis across tissues. High-resolution respirometry is the gold-standard measurement of mitochondrial OXPHOS and ET. The application of this method in pre-clinical and clinical cancer research has the capacity to expand current in vitro investigations to ex vivo studies. Furthermore, it offers potential applications in clinical and diagnostic settings.

All cells are intimately linked by their need to produce and consume adenosine triphosphate (ATP), the molecular energy currency. As cellular mutations give rise to the formation of tumors, mitochondria ensure survival through diversification of energy production that is often phenotypically distinguishable from non-cancerous tissue1,2,3. As such, there is a critical need for rapid and deep profiling of mitochondrial respiratory function in order to facilitate&#160;the classification of tumor type, cancer initiation, progression, and treatment response.
Respiratory functions of excised tissue specimens cannot be evaluated intact as the primary substrates for OXPHOS are not cell-permeable. To overcome this limitation, mitochondria can be prepared either by isolation, chemical permeabilization, or mechanical homogenization. Mitochondrial isolation is long considered to be a gold standard for the evaluation of respiratory function. However, it requires large amounts of tissue, is time-consuming, and is low-yielding with possible selection bias for certain fractions of mitochondria4. Permeabilization consists of mechanical separation and exposure of tissue sections or fiber bundles to a mild detergent that selectively degrades the plasma membrane5. Permeabilization is frequently employed in striated tissues such as skeletal and cardiac muscle as individual fiber bundles can be teased apart. Compared to isolation, permeabilization yields more mitochondria in their native cellular environment and physical form5. Permeabilization has been successfully applied in other tissues such as tumor6,7 and placenta8; however, reproducibility of permeabilized fiber preparations can be difficult due to consistency of dissection and oxygen requirements to overcome diffusion limitations9. Additionally, permeabilized fibers may be unsuitable in certain tumor types that are densely cellular and highly fibrotic. Tissue homogenates are generated through mechanical disruption of the plasma membrane and are similar to permeabilized fibers in terms of mitochondrial yield and integrity10. Tissue homogenates also minimize the limitations of oxygen diffusion and can be readily employed across tissue types through optimization of mechanical force11,12.
Here, we outline a simple and rapid method to quantify rates of oxidative phosphorylation (OXPHOS) and electron transfer (ET) capacity in freshly excised solid tumor homogenates. The protocol is optimally designed to evaluate fresh tissue using the Oxygraph-2k (O2k) high-resolution respirometer, which requires prior knowledge of instrumental setup and calibration but can be similarly adapted using any Clark-type electrode, Seahorse analyzer, or plate reader. The protocol can be reproducibly applied across species and tumor types as well as adapted to evaluate a diversity of mitochondrial ET pathways.

<table><tbody><tr><td>2-(N-Morpholino)ethanesulfonic acid hydrate</td><td>Sigma-Aldrich</td><td>M8250</td><td></td></tr><tr><td>Adenosine 5&amp;prime;-diphosphate sodium salt</td><td>Sigma-Aldrich</td><td>A2754</td><td></td></tr><tr><td>Adenosine 5&#039;-triphosphate disodium salt hydrate</td><td>Sigma-Aldrich</td><td>A2383</td><td></td></tr><tr><td>Amphotericin B</td><td>Gibco</td><td>15290018</td><td></td></tr><tr><td>Antimycin A</td><td>Sigma-Aldrich</td><td>A8674</td><td></td></tr><tr><td>Ascorbate</td><td>Sigma-Aldrich</td><td>A4544</td><td></td></tr><tr><td>Bovine serum albumin, fraction V, heat shock, fatty acid free</td><td>Sigma-Aldrich</td><td>3117057001</td><td>Roche</td></tr><tr><td>BD 50 mL Luer-Lok Syringe</td><td>Fisher Scientific</td><td>13-689-8</td><td></td></tr><tr><td>BD&amp;nbsp;Vacutainer General Use Syringe Needles</td><td>Fisher Scientific</td><td>23-021-020</td><td></td></tr><tr><td>Calcium carbonate</td><td>Sigma-Aldrich</td><td>C4830</td><td></td></tr><tr><td>Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone</td><td>Sigma-Aldrich</td><td>C2920</td><td></td></tr><tr><td>Cytochrome c from equine heart</td><td>Sigma-Aldrich</td><td>C2506</td><td></td></tr><tr><td>Datlab 7.4 software</td><td>Oroboros Instruments</td><td></td><td></td></tr><tr><td>Dimethylsulfoxide</td><td>Amresco</td><td>N182</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>D0632</td><td></td></tr><tr><td>D-Sucrose</td><td>Sigma-Aldrich</td><td>S7903</td><td></td></tr><tr><td>Dumont # 5 Forceps</td><td>Fine Science Tools</td><td>11251-30</td><td>Dumoxel, autoclavable</td></tr><tr><td>Dumont # 7 Forceps</td><td>Fine Science Tools</td><td>11271-30</td><td>Dumoxel, autoclavable</td></tr><tr><td>Digital Calipers 150 mm/6 in</td><td>World Precision Instruments</td><td>501601</td><td></td></tr><tr><td>EO771 cells</td><td>CH3 BioSystems</td><td>SKU:&amp;nbsp;94APV1-vial-prem</td><td>Pathogen Tested</td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-&lt;em&gt;N,N,N&amp;prime;,N&amp;prime;&lt;/em&gt;-tetraacetic acid</td><td>Sigma-Aldrich</td><td>E4378</td><td></td></tr><tr><td>Female C57BL/6J mice</td><td>&amp;nbsp;Jackson Laboratory</td><td>Stock #000664</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H4034</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>56750</td><td></td></tr><tr><td>Kimwipes</td><td>Fisher Scientific</td><td>34120</td><td></td></tr><tr><td>L-(&amp;minus;)-Malic acid</td><td>Sigma-Aldrich</td><td>G1626</td><td></td></tr><tr><td>Lactobionic acid</td><td>Sigma-Aldrich</td><td>L2398</td><td></td></tr><tr><td>Malate</td><td>Sigma-Aldrich</td><td>M6413</td><td></td></tr><tr><td>Matrigel Matrix</td><td>Corning</td><td>354248</td><td></td></tr><tr><td>MgCl&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M2670</td><td></td></tr><tr><td>Microsyringes</td><td>Hamilton</td><td>87919, 80383, 80521, 80665, 80765, 80865, 87943</td><td></td></tr><tr><td>N,N,N&amp;prime;,N&amp;prime;-Tetramethyl-p-phenylenediamine</td><td>Sigma-Aldrich</td><td>T7394</td><td></td></tr><tr><td>Oxygraph-2k</td><td>Oroboros Instruments</td><td>10023-03</td><td></td></tr><tr><td>Oxygraph-2k FluoRespirometer</td><td>Oroboros Instruments</td><td>10003-01</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Phosphocreatine disodium salt hydrate</td><td>Sigma-Aldrich</td><td>P7936</td><td></td></tr><tr><td>Potassium hydroxide</td><td>Sigma-Aldrich</td><td>P1767</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>P5655</td><td></td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>21875034</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P5280</td><td></td></tr><tr><td>Succinate (disodium)</td><td>Sigma-Aldrich</td><td>W327700</td><td></td></tr><tr><td>Taurine</td><td>Sigma-Aldrich</td><td>T0625</td><td></td></tr><tr><td>Whatman Filter Paper, grade 5</td><td>Sigma-Aldrich</td><td>1005-090</td><td></td></tr><tr><td>Wheaton Tenbroeck Tissue Grinder, 7 mL</td><td>Duran Wheaton Kimble</td><td>357424</td><td></td></tr><tr><td>Straight Tip Micro Dissecting Scissors</td><td>Roboz</td><td>RS-5914SC</td><td></td></tr><tr><td>Non-Safety Scalpel No. 11</td><td>McKesson</td><td>1029065</td><td></td></tr><tr><td>BD Precision Glide Needle 27 G x 1/2</td><td>Becton, Dickinson and Company</td><td>305109</td><td></td></tr><tr><td>BD Precision Glide Needle 18 G x 1</td><td>Becton, Dickinson and Company</td><td>305195</td><td></td></tr><tr><td>BD 1mL Slip Tip Syringe</td><td>Becton, Dickinson and Company</td><td>309659</td><td></td></tr><tr><td>Pyrex Reusable Petri Dish, 60 mm</td><td>Thermo Fisher Scientific</td><td>316060</td><td></td></tr><tr><td>Rodent Very High Fat Diet,&amp;nbsp; 60% kcal from fat, 20% kcal from protein, and 20% kcal from carbohydrate</td><td>Research Diet</td><td>D12492</td><td></td></tr><tr><td>Pyrex Watch Glass, 100 mm</td><td>Thermo Fisher Scientific</td><td>S34819</td><td></td></tr></tbody></table>

analytical determination of mitochondrial function of excised solid tumor homogenates

Analytical Determination of Mitochondrial Function of Excised Solid Tumor Homogenates

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule synthesis. Genetic and functional adaptations of mitochondria to the tumor environment drive proliferative and metastatic potential. The advent of DNA and RNA sequencing removed critical barriers to the evaluation of genetic mediators of tumorigenesis. However, to date, methodological approaches to evaluate tumor mitochondrial function remain elusive and require technical proficiency limiting the feasibility, ultimately diminishing diagnostic and prognostic value in both experimental and clinical settings. Here, we&#160;outline a simple and rapid method to quantify rates of oxidative phosphorylation (OXPHOS) and electron transfer (ET) capacity in freshly excised solid tumor homogenates using high-resolution respirometry. The protocol can be reproducibly applied across species and tumor types as well as adapted to evaluate a diversity of mitochondrial ET pathways. Using this protocol, we demonstrate that mice bearing a luminal B mammary cancer exhibit defective nicotinamide adenine dinucleotide-linked respiration and reliance on succinate to generate adenosine triphosphate via OXPHOS.

Initial studies revealed that EO771 tumors were lowly oxidative and thus required high homogenate concentrations for adequate O2 flux assessment. Optimization experiments were conducted to determine the optimal tissue homogenate concentration range for the study. Tumor homogenates were initially prepared at 40 mg/mL and then linearly diluted. The O2 flux normalized to tissue mass was consistent across concentrations (Figures 1A-D). It was observed that 40 mg/mL resulted in rapid oxygen depletion and was not suitable for experimentation (Figure 1A). The oxygen consumption slowed substantially with 30 mg/mL and 20 mg/mL but still decreased rapidly in a short time in the absence of substrates or ADP (Figure 1B,C). The 10 mg/mL concentration resulted in the optimum oxygen consumption rate (Figure 1D) that would support a longer, 90-min SUIT protocol.
A SUIT protocol was used to evaluate NADH- and succinate-linked OXPHOS and ET, as well as CIV activity (Figure 2A). Pyruvate and malate were added to the tissue homogenate in the absence of ADP to drive leak (L) through NADH. Saturating ADP was then added to drive maximal NADH-linked OXPHOS (P), followed by the addition of glutamate. Cytochrome c was then added to ensure outer membrane integrity; the increase in respiration rate was less than 20% in all samples (Figure 2B). Given the very low response to NADH-linked substrates, cytochrome c release was also assessed in the presence of succinate and rotenone and observed minimal cytochrome c stimulation (Figure 2B). Interestingly, NADH-linked OXPHOS was negligible in EO771 tumors (Figure 2C). Succinate was then added in the presence of pyruvate, malate, and glutamate to stimulate electron flow through succinate dehydrogenase. FCCP was then titrated to drive maximal electron flow (E), which revealed that in EO771 tumors, phosphorylation rather than oxidation was limiting to respiration (Figure 2C). Rotenone and antimycin A were subsequently titrated to inhibit complex I and complex III, respectively. Ascorbate and TMPD were then added to drive maximal electron flow through CIV, which is then inhibited by sodium azide. Table 1 illustrates analytical reduction equations of the raw data (Table 2) to quantitate the respiratory parameters plotted in Figure 2C. Overall, the tumor homogenate respiratory profiles (Figure 2C) are similar to those of non-implanted digitonin-permeabilized EO771 cells (Figure 2D) with the exception of diminished maximal electron transfer supported by N- and S-linked substrates in the tumor.
Since NADH-linked respiration was negligible, the respiratory kinetics of succinate were further evaluated by stepwise titrations of sub-saturating ADP until the maximal rate (VMAX) was achieved (Figure 3A,3B). The half-maximal concentration (KM) of ADP in the presence of succinate + rotenone was 37.5 µM, whereas the VMAX was ~10.5 pmol/s/mg (Figure 3C). Thus, despite relatively poor oxidation rates, EO771 tumors were highly sensitive to ADP and sustained ATP synthesis at relatively low ADP concentrations.
Selecting appropriate regions of the raw data for extraction is critical for the reproducibility of experiments and accurate quantification. For cytochrome c, a mark needs to be selected at the steady-state immediately prior to injection (Figure 4A, mark 1). There is often an initial injection artifact that can be followed by a period of time (about 5-10 min) where the O2 flux is not steady. Evaluation of cytochrome c efficiency is made by making an additional selection once the O2 flux has stabilized (Figure 4A, mark 2). Selections after the addition of substrates, ADP, or most inhibitors are also made after the injection artifact and once the O2 flux has stabilized (Figure 4B). The selection used to determine maximal uncoupled respiration is made at the peak increase achieved during titration of FCCP, which is often not the last injection made (Figure 4C). The selection for TMPD is made after both ascorbate and TMPD have been added and at the peak increase in respiration (Figure 4D, mark 1). Just after this peak, the inhibitor, sodium azide, is added, which rapidly decreases respiration but also often has an injection artifact lower than the inhibited respiration rate (Figure 4D). The inhibitor mark is made immediately after the injection artifact (Figure 4D, mark 2). The O2 flux will typically not stabilize and continue to decrease.
Table 1: Respiratory notation and analytical derivation. <a href="https://www.jove.com/files/ftp_upload/62875/TABLE%201.docx" target="_blank">Please click here to download this Table.</a>
Table 2: Sample and respiratory characteristics of luminal B mammary tumor homogenates. <a href="https://www.jove.com/files/ftp_upload/62875/TABLE%202.docx" target="_blank">Please click here to download this Table.</a>
<img alt="O₂ flux vs. concentration graph, analysis of O₂ consumption at varying concentrations over time." class="xfigimg" src="/files/ftp_upload/62875/62875fig01.jpg" data-altupdatedat="1788282455000" data-alt="Figure 1"> 
Figure 1: Optimization of tumor homogenate concentration. O2 Flux (red) and O2 Concentration (blue) in mammary tumor homogenates prepared at (A) 40 mg/mL, (B) 30 mg/mL, (C) 20 mg/mL, and (D) 10 mg/mL. Thom: Tissue homogenate respiration. <a href="https://www.jove.com/files/ftp_upload/62875/62875fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Oxygen flux analysis; O₂ flux/flow graphs; cytochrome c release; experimental comparison." class="xfigimg" src="/files/ftp_upload/62875/62875fig02.jpg" data-altupdatedat="1788282455000" data-alt="Figure 2"> 
Figure 2: Evaluation of OXPHOS and ET capacity by high-resolution respirometry in freshly excised tumor homogenates. (A) Representative plot of oxygen consumption (red) and concentrations (blue) over the course of a substrate, inhibitor, uncoupler protocol. PM: Pyruvate + Malate, D: ADP, G: Glutamate, c: Cytochrome c, S: Succinate, F: FCCP, Rot: Rotenone, Ama: Antimycin A, Asc/TMPD: Ascorbate/Tetramethyl-p-phenylenediamine. (B) Percent increase in O2 flux upon addition of cytochrome c. (C-D) Respiration supported by malate, pyruvate, glutamate, and succinate in the presence of ADP, FCCP, and ascorbate/TMPD in (C) EO771-derived tumor homogenates and (D) non-implanted EO771 digitonin-permeabilized cells. Thom: Tissue homogenate respiration; PM: Pyruvate + Malate; PMG: Pyruvate + Malate + Glutamate; PMGS: Pyruvate + Malate + Glutamate + Succinate; CIV: Complex IV; -L: Leak state; -P: Oxidative phosphorylation state, -E: Electron transfer state; N-linked: O2 flux supported by defined NADH-generating substrate combinations; NS-linked: O2 flux supported by the convergence of defined NADH-generate substrate combinations and succinate. <a href="https://www.jove.com/files/ftp_upload/62875/62875fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Mitochondrial respiration graph, O2 flux vs. ADP; kinetics plot and histogram; research data." class="xfigimg" src="/files/ftp_upload/62875/62875fig03.jpg" data-altupdatedat="1788282455000" data-alt="Figure 3"> 
Figure 3: EO771 mammary tumors displayed high ADP (adenosine 5′-diphosphate) sensitivity. (A) Representative plot of oxygen consumption (red) and concentrations (blue) throughout an S-linked ADP titration protocol. Thom: Tissue homogenate respiration; S/Rot: Succinate/Rotenone; D: ADP. (B) Respiration supported by succinate in the presence of rotenone and increasing concentrations of ADP (0 µM ADP = S/Rot-L). (C) Maximal rate (VMAX) and half-maximal concentration (KM) of ADP in the presence of succinate + rotenone. <a href="https://www.jove.com/files/ftp_upload/62875/62875fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="O₂ flux vs. concentration graph; oxygen consumption study; temporal variation analysis, experimental data." class="xfigimg" src="/files/ftp_upload/62875/62875fig04.jpg" data-altupdatedat="1788282455000" data-alt="Figure 4"> 
Figure 4: Representative tracing illustrating mark selection of raw O2 fluxes for data extraction. (A) Cytochrome c selection: selection number 1 before the Cytochrome c injection and selection number 2 after the injection when the O2 flux has stabilized. c Cytochrome c. (B) Substrate, ADP, and inhibitor selection: selection number 1 after the injection (succinate in this representative plot) where the O2 flux has stabilized. S: Succinate. (C) Uncoupler selection: selection number 1 at the peak increase in respiration during the uncoupler titration. In this representative FCCP titration plot, the third injection slightly decreases respiration and thus is not used for selection. F: ACCP. (D) TMPD selection: selection number 1 at the peak increase of respiration after the ascorbate and TMPD injections. Sodium azide selection: selection number 2 after the acute injection artifact when the respiration initially decreases. As/Tm: Ascorbate/TMPD; Azd: Azide. <a href="https://www.jove.com/files/ftp_upload/62875/62875fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

We developed a practical protocol and analytical approach to evaluate mitochondrial oxidative phosphorylation and electron transfer capacity in fresh tumor homogenates. This protocol can be easily adapted to survey various mitochondrial functions that contribute to cancer initiation, progression, and treatment response.

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule ...

This protocol describes a simple and rapid method for measuring tumor mitochondrial function. The protocol is broadly applicable to clinical translational oncology, and will help enhance diagnostic and mechanistic interpretation of the role of mitochondria in the onset and progression of cancer. The main advantage of this technique is the application of tissue homogenization to simplify preparation and maximize the yield of mitochondria while mitigating the limitations of diffusion.This technique offers potential applications in clinical and diagnostic settings with a quantitative assessment of tumor mitochondrial function using freshly biopsied or excised tumor tissue. Begin by placing a glass homogenizer containing one milliliter of mitochondrial respiration medium, or MiR05, in a tightly-fitting glass pestle on wet ice. Place the tissue in one milliliter of biopsy preservation solution, or BIOPS, in an ice-cold Petri dish.Cut the tumor into small pieces of approximately 5 to 10 milligrams each, and place the remaining tumor pieces back into 10 milliliters of BIOPS kept on ice. After blotting the tissue sections carefully on a filter paper, place them on a small plastic tared weigh boat and record the initial wet weight. Place the unused pieces back into the 10-milliliter conical tube of BIOPS for continued preservation.Submerge the tissue sections into an ice cold homogenizer containing MiR05 and record the remaining weight on the weigh boat. Using a glass pestle with a clearance range of 0.09 to 0.16 millimeters, gently disrupt the tumor tissue by completing five to seven down and up strokes. For each stroke, rotate the pestle in a clockwise/counter-clockwise motion three times while pushing the pestle down, and three additional times while pulling the pestle back up.Allow the tissue to settle to the bottom of the homogenizer in between strokes, but avoid bringing the pestle fully above the fluid volume to prevent foaming. Pour the homogenate into a 15-milliliter conical tube and place it on ice. Pipette one to three milliliters of fresh MiR05 over the pestle and into the homogenizer to wash any remaining tissue homogenate.Pour the MiR05 wash into the conical tube containing the homogenate. Repeat this step two to three times to ensure complete transfer of the homogenate. To accurately calculate the tissue homogenate concentration, carefully inspect the homogenizer and the homogenate for remaining non-homogenized material, which includes connective tissue.To remove the non-homogenized material from the homogenizer that cannot be reached with tweezers, after adding MiR05 to the homogenizer, aspirate the volume, including the tissue, with a pipette and move the contents to a Petri dish. Remove the non-homogenized material settled in large pieces at the bottom of the conical tube from the homogenate by aspirating them with a pipette, and place them on the cap of the conical tube. Remove the tissue from the Petri dish or conical tube cap with tweezers, and blot it on filter paper.Add the remaining homogenate from the conical tube cap back into the conical tube. Cap the tube, then invert to mix. Reinspect the homogenizers and homogenate for any additional non-homogenized material, and repeat the steps to remove non-homogenized material if needed.Weigh and record the mass of the non-homogenized material recovered from the homogenizer. Inspect the homogenate preparation for any grossly-intact tissue transferred, and removed the non-homogenized pieces and weigh as necessary. Subtract the tissue weight recovered from the weigh boat, homogenizer, and homogenate from the initial wet weight to calculate the final sample weight.Using the final sample weight, add additional MiR05 to bring homogenate to the desired concentration. Once the homogenate is weighed and prepared, proceed to assay as soon as possible. Keep the sample stored on wet ice until it is transferred to the instrument.Once the instrument is calibrated and the sample is prepared, remove the stoppers with a twisting motion and aspirate the MiR05 from the chambers, avoiding the membrane exposed inside the chamber. Mix the homogenate well, and add 2.25 milliliters of the homogenate to the chamber. Suppose one homogenate is being added to multiple chambers, pipette one milliliter of the homogenate at a time into each chamber while mixing the homogenate to ensure equal tissue distribution.Click on F4 to name and timestamp the event, and then click on Okay. Fill a 50-milliliter syringe with oxygen from an oxygen tank with a regulator and gas tubing using a blunt 18-gauge needle. Inject the oxygen directly into the chambers to hyperoxygenate them to approximately 500 micromolar oxygen.Loosely insert the stoppers and wait to close until the oxygen reaches approximately 480-micromolar. With a twisting motion, slowly close the chamber and allow the respiration to equilibrate for approximately 15 to 20 minutes. Fill the stopper's central capillary with MiR05 if necessary.Use dedicated microsyringes to inject substrates, uncouplers, and inhibitors into fully closed chambers to name and timestamp the events for each chamber in real time. Select F6 to adjust the oxygen concentration and oxygen slope negative scales as needed. After each injection, wash the syringe three times in water for water-soluble compounds, and 70%ethanol for ethanol-dissolved compounds.Add five microliters of 0.8-molar malate and proceed immediately to the next injection. Wash the injection syringe three times in water. Immediately add five microliters of 1-molar pyruvate, and wait for the respiration to stabilize.After washing the syringe, add 10 microliters of 0.5-molar ADP and wait for the ADP response to stabilize. Wash the syringe and add five microliters of 2-molar glutamate and wait for the respiration to stabilize. Wash the syringe, then add five microliters of 4-millimolar cytochrome-C, and wait for the respiration to stabilize.After washing the syringe, add 20 microliters of 1-molar succinate and wait for the respiration to stabilize. Titrate 0.5-to 1-microliter increments of 1-millimolar FCCP and wait for the respiration to stabilize after each injection. Continue until there is no additional increase in respiration.Wash the injection syringe three times in 70%ethanol. Add two microliters of 150-micromolar rotenone and wait for the respiration to stabilize. Add another one microliter of rotenone to ensure there is no further inhibition.If there is a decrease in respiration, continue with additional injections until there is no decrease in respiration, then wash the injection syringe three times in 70%ethanol. Add two microliters of 125-micromolar antimycin A and wait for the respiration to stabilize. Add another one microliter of antimycin A to ensure there is no further inhibition.If there is a decrease in respiration, continue with additional injections until there is no decrease in respiration. Wash the syringe three times with 70%ethanol. Check the oxygen concentration of the chamber.If the concentration is below 125-micromolar, reoxygenate to room air, or mildly hyperoxygenate to ensure that oxygen does not limit the respiratory flux. Add five microliters of 0.8-molar ascorbate. Wash the syringe three times in 70%ethanol.Immediately add 10 microliters of 0.2-molar TMPD, and wait for an increase in respiration. After washing the syringe with ethanol, add 25 microliters of 4-molar sodium azide immediately when the respiratory flux of ascorbate TMPD plateaus. End the by clicking on File, Save and disconnect.It was observed that 40 milligrams per milliliter of tissue homogenate resulted in rapid oxygen depletion. The oxygen consumption slowed substantially with 30 and 20 milligrams per milliliter of tissue homogenate, but still decreased rapidly in a short time in the absence of substrates or ADP. The 10 milligrams per milliliter concentration of tissue homogenate resulted in the optimum oxygen consumption rate.A SUIT protocol was used to evaluate NADH-and succinate-linked oxidative phosphorylation and electron transfer, as well as Complex IV activity. Cytochrome C release was assessed in the presence of pyruvate and malate, or succinate and rotenone, both of which revealed that the homogenate preparation did not damage the mitochondria. It was also found that NADH-linked oxidative phosphorylation was negligible in EO771 tumors.Titration of FCCP to drive maximal electron flow revealed that in EO771 tumors, phosphorylation, rather than oxidation, was limiting to respiration. The tumor homogenate respiratory profiles were similar to those of non-implanted, digitonin-permeabilized EO771 cells, except for diminished maximal electron transfer supported by N-and S-linked substrates in the tumor. The respiratory kinetics of succinate were further evaluated by stepwise titrations of subsaturating ADP until the maximal rate was achieved.The half-maximal concentration of ADP in the presence of succinate and rotenone was 37.5-micromolar, whereas the maximal rate was approximately 10.5 picomoles per second, per milligram. Instruments set up in routine care, as well as tissue handling, are of critical importance for the success of these experiments. Following these procedures, other mass-or marker-specific approaches to rate normalization can be applied based upon the question of interest.Common measures include total protein quantification and enzymatic activity of citrate synthase, but vary based upon the model system, statistical design, and research question.

analytical-determination-mitochondrial-function-excised-solid-tumor

Research

JoVE Journal

Cancer Research

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &lt;30 min compared to 60 - 100 min using alternative methods.

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

Scopri un metodo rapido per misurare la funzione mitocondriale nei tumori solidi usando omogeneizzati di tessuto fresco. Questa tecnica semplifica la preparazione campionaria e massimizza la resa mitocondriale per applicazioni cliniche.

Analytical Determination of Mitochondrial Function of Excised Solid Tumor Homogenates

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