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.

<ol>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-(N-Morpholino)ethanesulfonic acid hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M8250</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-diphosphate sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>A2754</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-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&#160;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:&#160;94APV1-vial-prem</td>
			<td>Pathogen Tested</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Female C57BL/6J mice</td>
			<td>&#160;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-(&#8722;)-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&#183;6H2O</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&#8242;,N&#8242;-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,&#160; 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

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&#160;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)&#160;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 &#181;M, 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 1.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 2.docx" target="_blank">Please click here to download this Table.</a>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62875/62875fig01.jpg" /> 
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="Figure 2" class="xfigimg" src="/files/ftp_upload/62875/62875fig02.jpg" /> 
Figure 2: Evaluation of OXPHOS and ET capacity by high-resolution respirometry in freshly excised tumor homogenates. (A)&#160;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&#160;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="Figure 3" class="xfigimg" src="/files/ftp_upload/62875/62875fig03.jpg" /> 
Figure 3: EO771 mammary tumors displayed high ADP (adenosine 5&#8242;-diphosphate) sensitivity. (A)&#160;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 &#181;M 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="Figure 4" class="xfigimg" src="/files/ftp_upload/62875/62875fig04.jpg" /> 
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>

Watch this Scientific Journal Video about Analytical Determination of Mitochondrial Function of Excised Solid Tumor Homogenates at JoVE.com

Analytical Determination of Mitochondrial Function of Excised Solid Tumor Homogenates

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 ...

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

Research

JoVE Journal

Cancer Research

2.5K Views.  Pennington Biomedical Research Center. 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.

Video: Analytical Determination of Mitochondrial Function of Excised Solid Tumor Homogenates

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

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Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate.
Extracellular flux (XF) analyzers have been used to explore mitochondrial function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondrial energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers,&#160;DNA content and type.
The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.

These protocols will help users probe mitochondrial energy metabolism in 3D cancer cell-line-derived spheroids using Seahorse extracellular flux analysis.

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing ...