We would like to acknowledge research support from the Department of Defense Prostate Cancer Research Program Award W81XWH-20-1-0274 and Prostate Cancer Foundation Challenge Award 16CHAL13. We would like to thank and acknowledge Dr. Michelle Rudek, Dr. Noushin Rastkari, Dr. Nicole Anders, and Linping Xu of the Analytical Pharmacology Shared Resource at Johns Hopkins for assistance with equol LC/MS/MS.

All animal studies were performed following institutional guidelines and under Johns Hopkins University Institutional Animal Care and Use Committee (IACUC) approved protocols M021M197 and M023M195. Mouse strains used (male mice, 7 weeks old) are described in the Table of Materials. The use of male mice was due to their use in the ongoing studies of prostate cancer. The MDA method has previously been shown to be effective in female mice as well7,17. Mice were randomized to their respective cages/treatment groups. The mice were treated in cages and in no particular order. The experimenter administering the treatments was blinded to the treatment group. The number assigned to a mouse referred to their ID and not the order in which they were treated. The data was collected throughout the timeline of the study and analyzed at the end to avoid any bias. Experimental assays were blinded to the treatment group until all data was collected.
NOTE: This protocol has been modified from Scarborough et al.7.
1. Preparation of treatments
<ol>
	<li>Prepare a solution of sweetened condensed milk (ingredients: milk and sugar) and molecular biology grade water using a 3:10 ratio of milk to water. Measure the sweetened condensed milk using a 15 mL conical tube due to the viscosity of the milk.</li>
	<li>Using a 50 mL conical tube, mix molecular biology-grade water with the sweetened condensed milk by slowly mixing 5 mL of water at a time with the milk. 
	NOTE: This step is performed under sterile conditions using a biosafety cabinet.</li>
	<li>Prepare aliquots of the milk solution (stored as 500 &#181;L aliquots) and store the aliquots at 4 &#176;C until combined with the treatment of interest.</li>
	<li>Resuspend treatments in their respective vehicles at the correct concentration based on the animal&#39;s body weight before combining them with the sweetened condensed milk/water solution.</li>
</ol>
2.&#160;MDA training session
NOTE: This section describes mouse models; however, the MDA technique could be scaled up as needed with larger volumes/pipette tips for larger rodent models.
<ol>
	<li>OPTIONAL: Gently restrain the study animal by holding the scruff of the neck with one hand.</li>
	<li>Offer the study animal 100 &#181;L of sweetened condensed milk/water solution only (without the addition of the experimental treatment) using a single channel pipette and a 200 &#181;L pipette tip which is guided to the mouth.</li>
	<li>Dispense the milk/water solution slowly to avoid missing the animal&#39;s mouth. Ensure the animal readily consumes the milk solution between 5-15 s. 
	NOTE: This training occurs for a minimum of 2 consecutive days (one training session per day), with no changes to the volume administered. A gentle restraint by scruff was needed for the MDA training sessions. Scarborough et al.7 recommend full restraint of the animal on the first day of MDA training and then restraint by the tail on the second day. The method of restraint may differ for larger rodent models. In contrast, Vanhecke et al.17 only held the mice slightly by the tail during training.</li>
</ol>
3. Mouse treatments using the MDA method
<ol>
	<li>Combine the study treatment with the sweetened condensed milk/water solution (see examples with validation experiments [section 4 and section 5] below).</li>
	<li>OPTIONAL: Gently restrain the study animal by holding the scruff of the neck with one hand. 
	NOTE: Scarborough et al.7 report that mice no longer require restraint beyond the training period and will readily drink from the pipette tip. In contrast, Vanhecke et al.17 report that for some treatments (e.g., tamoxifen) that might have mild taste aversion, MDA is best performed by gently restraining the mice via scruff during all treatments. This study also determined that positioning the animal on their back with gentle scruff improves the time and efficacy of treatment consumption.</li>
	<li>Administer 100 &#181;L of treatment using a single channel pipette and a 200 &#181;L pipette tip guided to the animal&#39;s mouth, as performed during the training sessions.</li>
	<li>Treat a single animal at a time.</li>
	<li>Place each animal back into their respective cage after treatment. 
	NOTE: Treatments using the MDA method can be given daily or in a repeated fashion7,17.</li>
</ol>
4. Validation experiment #1 - oral delivery of gut bacteria to mice using MDA
NOTE: This section describes the use of the MDA method to orally deliver live bacteria for gut colonization studies in mice. In this representative pilot experiment, the gram-positive bacterium Clostridium scindens is delivered to C57BL/6J mice. The mice were treated with antibiotics (cefoxitin) for two consecutive days in the drinking water prior to bacterial inoculation with MDA.
<ol>
	<li>Bacterial culture
	<ol>
		<li>Culture C. scindens strain ATCC 35704 in reinforced clostridial broth (RCB) using a frozen glycerol stock to inoculate 500 &#181;L of bacteria into a 7 mL RCB tube. Grow anaerobically overnight at 37 &#176;C.</li>
		<li>Use 500 &#181;L of the overnight bacteria culture to inoculate a new RCB tube and incubate anaerobically for 24 h. Remove this tube from the anaerobic chamber and use it to prepare the bacterial treatment for MDA. Use this same tube to calculate the colony-forming units per milliliter (CFU/mL).</li>
	</ol>
	</li>
	<li>Calculating C. scindens CFU/mL
	<ol>
		<li>Create a 1:3 dilution of the C. scindens culture using 3.5 mL of the culture inoculated into 7 mL of RCB media. Use a sample of this dilution to make a 20% 1 mL glycerol stock (1:1) that is stored at -80 &#176;C.</li>
		<li>Plate 100 &#181;L of the 1:3 dilution onto reinforced clostridial agar (RCA) plates. Use the first dilution to then make subsequent serial dilutions and plate for a total of three more times.</li>
		<li>After incubation of the RCA plates, count the colonies and calculate the CFU. Use the following equation to calculate the concentration of bacterial cells in the diluted and original samples: 
		CFU in diluted sample (cells/mL) = (number of colonies counted on the RCA plate)/(amount of diluted sample added to the RCA plate in mL) 
		&#8203;CFU in original sample (cells/mL) = (CFU in diluted sample)/(dilution of the RCA plate)</li>
	</ol>
	</li>
	<li>Preparing the C. scindens treatment for MDA
	<ol>
		<li>Combine a solution of 50 &#181;L (1.265 x 106 CFU/mL in this example) of C. scindens + 50 &#181;L of the sweetened condensed milk/water solution. 
		NOTE: If a specific CFU is needed, an alternative method of bacterial cell quantification may be used, such as creating a standard curve with optical density (OD) measurements at 600 nm.</li>
	</ol>
	</li>
	<li>Oral treatment of mice with C. scindens using MDA
	<ol>
		<li>On days 1-3, familiarize the mice with the milk solution as described in section 2 (MDA training session).</li>
		<li>On day 4, administer 100 &#181;L of the C. scindens + milk solution or PBS (control) + milk solution via MDA to C57BL/6J mice. Treat mice once for the duration of the study.</li>
	</ol>
	</li>
	<li>Fecal sample collection
	<ol>
		<li>Hold the mouse by gentle scruff and wait for them to discharge a fecal pellet. Collect fecal pellets directly from the rectum into a sterile 1.5 mL microcentrifuge tube. Store the samples at -80 &#176;C until use.</li>
	</ol>
	</li>
	<li>C. scindens quantitative PCR (qPCR)
	<ol>
		<li>Perform fecal DNA extraction using a previously published protocol19,20.</li>
		<li>Determine DNA concentration using a DNA fluorometer for high-sensitivity detection. Normalize DNA samples to 10 ng/&#181;L and store at -20 &#176;C until use.</li>
		<li>Use qPCR to investigate the abundance of: (i) C. scindens bacteria using conserved primers against the desA gene of C. scindens strain ATCC 35704, (ii) total bacteria using primers designed against the V6 hypervariable region of the bacterial 16S rRNA gene as previously described20,21. 
		NOTE: The qPCR conditions and primers used are listed in Table 1. A standard curve using C. scindens strain ATCC 35704 DNA was used to estimate the copies of desA+ bacteria.</li>
	</ol>
	</li>
</ol>
5. Validation experiment #2 - experimental drug delivery to mice using MDA
NOTE: This section describes the use of the MDA method to deliver an experimental compound to mice. In this representative pilot experiment, the soy metabolite equol (S-equol) is delivered to NSG and FVB/NJ mice.
<ol>
	<li>Preparation of S-equol treatment 
	NOTE: Both S-equol and dimethyl sulfoxide (DMSO) are aliquoted and stored at -20 &#176;C until use.
	<ol>
		<li>Reconstitute S-equol in DMSO combined with corn oil (90%). Mix aliquoted S-equol in corn oil (220 &#181;L) with 480 &#181;L of the sweetened condensed milk/water solution. Combine and mix this solution in the animal room to avoid the separation of the oil from the milk. 
		NOTE: If separation of the solution is observed, mix the solution before treatment. The dose of S-equol administered is 25 mg/kg/day. The mice used in this study were all similar in weight, so they received the same dose. The dose of the treatment may need to be adjusted and made into multiple preparations based on the study animal weights.</li>
		<li>Combine DMSO vehicle with corn oil (90%). Mix aliquoted DMSO in corn oil (220 &#181;L) with 480 &#181;L of the sweetened condensed milk/water solution. Combine and mix this solution in the animal room to avoid the separation of the oil from the milk. 
		NOTE: If separation of the solution is observed, mix the solution before treatment.</li>
	</ol>
	</li>
	<li>Oral treatment of mice with S-equol using MDA
	<ol>
		<li>On days 1 and 2, familiarize the mice with the milk solution as described in section 2 (MDA training session). 
		NOTE: For validation experiment #1, the training time described in Scarborough et al. was followed, which suggested a 3-day acclimation time. In this experiment, a shorter training time (2 days), was tested and found that it did not alter the administration of the treatments of interest.</li>
		<li>On days 3-43, treat mice with (i) PBS control, (ii) DMSO vehicle, or (iii) S-equol, all combined with the sweetened condensed milk/water solution. Make a master mix of the solution to deliver a homogenous solution of the treatments to all study animals. Administer a total volume of 100 &#181;L to each mouse once daily, 5 days a week, for a total of 43 days.</li>
	</ol>
	</li>
	<li>Blood collection
	<ol>
		<li>Collect blood after euthanasia (carbon dioxide overdose) via heart puncture. Transfer blood to a microtainer serum separator tube, mix, and store at room temperature (RT) until further processing.</li>
		<li>Centrifuge blood sample to complete separation at 254 x g for 5 min. Collect serum (top layer of separation) and store at -20 &#176;C until use.</li>
	</ol>
	</li>
	<li>S-equol liquid chromatography with tandem mass spectrometry (LC/MS/MS) assay
	<ol>
		<li>Refer to the Table of Materials file for the chemicals and reagents used for this experiment.</li>
		<li>Calibration standards and quality control: Prepare stock solutions of S-equol and internal standard (racemic equol-d4) in DMSO at concentrations of 1 mg/mL and store at -20 &#176;C.</li>
		<li>Dilute the stock standard solutions with acetonitrile:water (50:50) to prepare a mixed working solution with different concentrations. Prepare internal standard in ethyl acetate as extraction solution at a concentration of 100 ng/mL. Store stock and working solutions at 4 &#176;C and use daily, diluted, or directly.</li>
		<li>Sample extraction
		<ol>
			<li>Prepare the sample using protein precipitation by extraction solution (ethyl acetate plus internal standard, see section 5.4.3), followed by centrifugation and evaporation of supernatant under dry nitrogen (50 &#176;C).</li>
			<li>Reconstitute the sample in 100 &#181;L of 50% acetonitrile and transfer to autosampler vials for LC/MS/MS analysis.</li>
		</ol>
		</li>
		<li>Sample separation
		<ol>
			<li>Achieve separation with a C18 column, 2.1x50 mm, 1.7 &#181;m. Use 0.1% formic acid as mobile phase A and 0.1% formic acid in acetonitrile mobile phase B.</li>
			<li>Use a gradient and hold mobile phase B at 40% with a flow rate of 0.3 mL/min for 0.5 min. Then, increase the flow rate to 100% over 1 min, hold there for 2 min, and then return to 40% B for 1 min.</li>
			<li>Monitor the column effluent using a mass-spectrometric detector using electrospray ionization, which is operating in negative mode. Program the spectrometer to monitor the following multiple reaction monitoring (MRM) transitions: 241 &#8594; 119.1 for S-equol and 245 &#8594; 123 for the internal standard, equol-d4.</li>
			<li>Compute the calibration curve for S-equol using the area ratio peak of the analysis to the internal standard by using a quadratic equation with a 1/x2 weighting function using the calibration ranges of 5-500 ng/mL.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
6. Measuring the effect of MDA treatment on core gut microbiota
NOTE: This section describes using qPCR to measure levels of core gut microbiota after MDA treatment.
<ol>
	<li>Perform fecal sample collection and DNA extraction as described in steps 4.5.1 and 4.6.1.</li>
	<li>Determine DNA concentration using a DNA fluorometer for high-sensitivity detection. Normalize DNA samples to 10 ng/&#181;L and store at -20 &#176;C until use.</li>
	<li>Use qPCR to investigate the abundance of members of the gut microbiota: (i) Akkermansia muciniphila, (ii) Bifidobacterium spp., (iii) Streptococcus salivarius, (iv) Lactobacillus spp., and (v) total bacteria using primers designed against the V6 hypervariable region of the bacterial 16S rRNA gene as previously described20,21,22. 
	NOTE: The qPCR conditions and primers used are listed in Table 2.</li>
</ol>

Reducing Research Animal Stress with Micropipette-Guided Drug Administration

<ol>
	<li>Vandenberg, L. N., Welshons, W. V., Vom Saal, F. S., Toutain, P. -. L., Myers, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Should+oral+gavage+be+abandoned+in+toxicity+testing+of+endocrine+disruptors.">Should oral gavage be abandoned in toxicity testing of endocrine disruptors.</a> Environ Health. 13 (1), 46 (2014).</li><li>Craig, M. A., Elliott, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+fed+radiolabeled+protein+by+gavage+show+sporadic+passage+of+large+quantities+of+intact+material+into+the+blood,+an+artifact+not+associated+with+voluntary+feeding.">Mice fed radiolabeled protein by gavage show sporadic passage of large quantities of intact material into the blood, an artifact not associated with voluntary feeding.</a> Contemp Top Lab Anim Sci. 38 (5), 18-23 (1999).</li><li>Turner, P. V., Brabb, T., Pekow, C., Vasbinder, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+substances+to+laboratory+animals:+routes+of+administration+and+factors+to+consider.">Administration of substances to laboratory animals: routes of administration and factors to consider.</a> J Am Assoc Lab Anim Sci. 50 (5), 600-613 (2011).</li><li>Gulin, J. E. N., Bisio, M., Garc&#237;a-Bournissen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refining+drug+administration+in+a+murine+model+of+acute+infection+with+Trypanosoma+cruzi.">Refining drug administration in a murine model of acute infection with Trypanosoma cruzi.</a> Lab Anim Res. 36, 37 (2020).</li><li>Bonnichsen, M., Dragsted, N., Hansen, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+welfare+impact+of+gavaging+laboratory+rats.">The welfare impact of gavaging laboratory rats.</a> Anim Welf. 14 (3), 223-227 (2005).</li><li>Brown, A. P., Dinger, N., Levine, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+produced+by+gavage+administration+in+the+rat.">Stress produced by gavage administration in the rat.</a> Contemp Top Lab Anim Sci. 39 (1), 17-21 (2000).</li><li>Scarborough, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+validation+of+the+micropipette-guided+drug+administration+(MDA)+method+in+the+maternal+immune+activation+model+of+neurodevelopmental+disorders.">Preclinical validation of the micropipette-guided drug administration (MDA) method in the maternal immune activation model of neurodevelopmental disorders.</a> Brain Behav Immun. 88, 461-470 (2020).</li><li>Germann, P. G., Ockert, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulomatous+inflammation+of+the+oropharyngeal+cavity+as+a+possible+cause+for+unexpected+high+mortality+in+a+Fischer+344+rat+carcinogenicity+study.">Granulomatous inflammation of the oropharyngeal cavity as a possible cause for unexpected high mortality in a Fischer 344 rat carcinogenicity study.</a> Lab Anim Sci. 44 (4), 338-343 (1994).</li><li>Martins, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+gelatin+flavors+for+oral+dosing+of+C57BL/6J+and+FVB/N+mice.">Comparison of gelatin flavors for oral dosing of C57BL/6J and FVB/N mice.</a> J Am Assoc Lab Anim Sci. 61 (1), 89-95 (2022).</li><li>Huynh, N., Arabian, N., Lieu, D., Asatryan, L., Davies, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+an+orally+dissolving+strip+for+pharmacological+and+toxicological+studies:+A+simple+and+humane+alternative+to+oral+gavage+for+animals.">Utilizing an orally dissolving strip for pharmacological and toxicological studies: A simple and humane alternative to oral gavage for animals.</a> J Vis Exp. 109, e53770 (2016).</li><li>Hoggatt, A. F., Hoggatt, J., Honerlaw, M., Pelus, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spoonful+of+sugar+helps+the+medicine+go+down:+a+novel+technique+to+improve+oral+gavage+in+mice.">A spoonful of sugar helps the medicine go down: a novel technique to improve oral gavage in mice.</a> J Am Assoc Lab Anim Sci. 49 (3), 329-334 (2010).</li><li>Neto, T., Faustino-Rocha, A. I., Gil da Costa, R. M., Medeiros, R., Oliveira, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quick+and+low-intensity+method+for+oral+administration+to+large+numbers+of+mice:+A+possible+alternative+to+oral+gavage.">A quick and low-intensity method for oral administration to large numbers of mice: A possible alternative to oral gavage.</a> Lab Anim. 56 (2), 185-190 (2022).</li><li>K&#252;ster, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voluntary+ingestion+of+antiparasitic+drugs+emulsified+in+honey+represents+an+alternative+to+gavage+in+mice.">Voluntary ingestion of antiparasitic drugs emulsified in honey represents an alternative to gavage in mice.</a> J Am Assoc Lab Anim Sci. 51 (2), 219-223 (2012).</li><li>Gonzales, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+method+of+oral+administration+by+peanut+butter+pellet+formulation+results+in+target+engagement+of+BACE1+and+attenuation+of+gavage-induced+stress+responses+in+mice.">Alternative method of oral administration by peanut butter pellet formulation results in target engagement of BACE1 and attenuation of gavage-induced stress responses in mice.</a> Pharmacol Biochem Behav. 126, 28-35 (2014).</li><li>Teixeira-Santos, L., Albino-Teixeira, A., Pinho, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+method+for+oral+drug+administration+by+voluntary+intake+in+male+and+female+mice.">An alternative method for oral drug administration by voluntary intake in male and female mice.</a> Lab Anim. 55 (1), 76-80 (2021).</li><li>Schalbetter, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+application+of+clozapine-N-oxide+using+the+micropipette-guided+drug+administration+(MDA)+method+in+mouse+DREADD+systems.">Oral application of clozapine-N-oxide using the micropipette-guided drug administration (MDA) method in mouse DREADD systems.</a> Lab Anim (NY). 50 (3), 69-75 (2021).</li><li>Vanhecke, D., Bugada, V., Buch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refined+tamoxifen+administration+in+mice+by+encouraging+voluntary+consumption+of+palatable+formulations.">Refined tamoxifen administration in mice by encouraging voluntary consumption of palatable formulations.</a> bioRxiv. , (2023).</li><li>Heraudeau, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropipette-guided+drug+administration+(MDA)+as+a+non-invasive+chronic+oral+administration+method+in+male+rats.">Micropipette-guided drug administration (MDA) as a non-invasive chronic oral administration method in male rats.</a> J Neurosci Methods. 398, 109951 (2023).</li><li>Sfanos, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compositional+differences+in+gastrointestinal+microbiota+in+prostate+cancer+patients+treated+with+androgen+axis-targeted+therapies.">Compositional differences in gastrointestinal microbiota in prostate cancer patients treated with androgen axis-targeted therapies.</a> Prostate Cancer Prostatic Dis. 21 (4), 539-548 (2018).</li><li>Shrestha, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+the+urinary+microbiome+in+men+with+positive+versus+negative+biopsies+for+prostate+cancer.">Profiling the urinary microbiome in men with positive versus negative biopsies for prostate cancer.</a> J Urol. 199 (1), 161-171 (2018).</li><li>Peiffer, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+of+gastrointestinal+microbiota+in+association+with+treatment+response+in+individuals+with+metastatic+castrate+resistant+prostate+cancer+progressing+on+enzalutamide+and+initiating+treatment+with+anti-PD-1+(pembrolizumab).">Composition of gastrointestinal microbiota in association with treatment response in individuals with metastatic castrate resistant prostate cancer progressing on enzalutamide and initiating treatment with anti-PD-1 (pembrolizumab).</a> Neoplasia. 32, 100822 (2022).</li><li>Jones, C. B., Peiffer, L. B., Davis, C. M., Sfanos, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+the+effects+of+4He+exposure+on+the+gut-brain+axis.">Examining the effects of 4He exposure on the gut-brain axis.</a> Radiat Res. 197 (3), 242-252 (2022).</li><li>Walker, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+less+stressful+alternative+to+oral+gavage+for+pharmacological+and+toxicological+studies+in+mice.">A less stressful alternative to oral gavage for pharmacological and toxicological studies in mice.</a> Toxicol Appl Pharmacol. 260 (1), 65-69 (2012).</li><li>Miguelena Chamorro, B., Swaminathan, G., Mundt, E., Paul, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+more+translatable+research:+Exploring+alternatives+to+gavage+as+the+oral+administration+route+of+vaccines+in+rodents+for+improved+animal+welfare+and+human+relevance.">Towards more translatable research: Exploring alternatives to gavage as the oral administration route of vaccines in rodents for improved animal welfare and human relevance.</a> Lab Anim (NY). 52 (9), 195-197 (2023).</li></ol>

OG can be a significant source of stress in research animals that may create a confounding variable as previously assessed in multiple studies7,9,11,12,13,14,15,23. Due to the invasiveness of OG, alternate techniques have been employed to minimize the chall...

Drug administration to rodent models is commonly achieved via oral gavage (OG), which consists of administering a liquid preparation directly to the stomach using a cannula attached to a syringe containing the solution. This technique results in a consistent and precise dosage of the treatment to the animal, but also carries multiple disadvantages. OG has been scrutinized for not adequately modeling human dietary exposures1,2. Furthermore, OG increases the risk of unintentional injuries to the upper digestive system (perforation of the esophagus and stomach), aspiration of the administered treatment, and respiratory tract lesions3. OG is also associated with discomfort4, increased blood pressure and heart rate5, as well as stress6,7, and sometimes death8 due to decreased tolerance to gavage by the animal. These physiologic changes might interfere with or confound experimental results; thus, new procedures have been explored to avoid these side effects. Studies have utilized alternative procedures to OG, such as the use of gelatin as a drug vehicle9, orally dissolving strips (ODS)10, sucrose-coated gavage needles11, flexible feeding tubes, wheat cookies12, honey13, and peanut butter pellets14. Unfortunately, there are limitations with these modifications to the OG technique, including incompatibility with water-insoluble drugs, longer preparation time for the treatment15, drug palatability, and stability15, and familiarization of the animal with the food. Furthermore, there is potential for less precise dosing when animals feed ad lib.
Scarborough et al.7 previously developed an alternative oral treatment method in mice, which they termed micropipette-guided drug administration (MDA). This method of administration is based on a sweetened condensed milk solution as a vehicle for pharmacological substances, motivating the study animals to consume the prepared vehicle and/or drug solutions readily via dispensing the solution with a single channel pipette and pipette tip. To introduce this technique, rodents undergo a training session (minimum 2 days) to shorten handling times and to allow the study animal to become familiar with drinking from the pipette tip4. Initial validation studies by Scarborough et al.7 and Schalbetter et al.16 suggest that the MDA procedure is easy to implement, cost-effective, minimally invasive, and less stressful for the animals than conventional oral gavage methods. Scarborough et al. introduced the use of the MDA technique in a mouse model of maternal immune activation (MIA) of neurodevelopmental disorders7. This study demonstrated that the pharmacokinetic profiles of mice treated with the antipsychotic drug risperidone using MDA were comparable to the use of OG. Furthermore, MDA did not induce an increase in corticosterone (a stress hormone) levels in the mice, and chronic treatment with risperidone using the MDA technique led to a dose-dependent decrease in MIA-induced social interaction deficits and amphetamine hypersensitivity7. Additional studies have explored the efficacy of MDA versus OG in both mouse17 and rat18 models. MDA has also been compared to intraperitoneal injection and was shown to be as effective in delivering clozapine-N-oxide to mice16. Due to MDA&#39;s reported success in reducing animal stress and therapeutic efficacy, we now aim to further explore the MDA technique as an effective method of drug delivery using additional murine models. Here, we describe the implementation of the MDA method to treat different mouse strains, including the immune-competent FVB/NJ and C57BL/6J strains and the immunocompromised NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) strain with different oral treatments, including live bacteria, experimental compounds delivered in water-insoluble solutions (corn oil), and vehicle controls. We assessed the activity and presence of the different treatments in serum and feces, and we evaluated alterations to core gut microbiota in relation to the MDA method.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>200 &#181;L pipette tips</td>
			<td>Mettler Toledo</td>
			<td>17005860</td>
			<td></td>
		</tr>
		<tr>
			<td>AB SCIEX Triple QTRAP 5500 mass-spectrometric detector</td>
			<td>Sciex</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Akkermansia muciniphila strain muc genomic DNA</td>
			<td>American Type Culture Collection</td>
			<td>BAA-835D-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium acetate</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>5.43834</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Jackson Laboratories</td>
			<td>Strain# 000664</td>
			<td></td>
		</tr>
		<tr>
			<td>C. scindens strain 35704</td>
			<td>American Type Culture Collection</td>
			<td>35704</td>
			<td></td>
		</tr>
		<tr>
			<td>Cefoxitin</td>
			<td>Sagent&#160;</td>
			<td>NDC25021-109-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Corn oil</td>
			<td>MedChemExpress</td>
			<td>HY-Y1888</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Fisher Scientific</td>
			<td>AC611050040</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>5.33002</td>
			<td></td>
		</tr>
		<tr>
			<td>FVB/NJ mice</td>
			<td>Jackson Laboratories</td>
			<td>Strain# 001800</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Fisher Scientific</td>
			<td>02-002-996</td>
			<td></td>
		</tr>
		<tr>
			<td>LC-MS grade water</td>
			<td>Fisher Scientific</td>
			<td>14-650-357</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>02-003-340</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtainer serum separator tube</td>
			<td>Becton Dickinson</td>
			<td>02-675-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular biology grade water</td>
			<td>Corning</td>
			<td>46-000-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>NSG mice</td>
			<td>Jackson Laboratories</td>
			<td>Strain# 005557</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit DNA HS kit</td>
			<td>Invitrogen</td>
			<td>Q32851</td>
			<td></td>
		</tr>
		<tr>
			<td>Racemic&#160;equol-d4</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-219827</td>
			<td></td>
		</tr>
		<tr>
			<td>Reinforced Clostridial agar</td>
			<td>Anaerobe Systems</td>
			<td>AS-6061</td>
			<td></td>
		</tr>
		<tr>
			<td>Reinforced Clostridial broth</td>
			<td>Anaerobe Systems</td>
			<td>AS-606</td>
			<td></td>
		</tr>
		<tr>
			<td>S-equol</td>
			<td>MedChemExpress</td>
			<td>HY-100583</td>
			<td></td>
		</tr>
		<tr>
			<td>S-equol reference standard for LC-MS</td>
			<td>Cayman Chemical</td>
			<td>10010173</td>
			<td></td>
		</tr>
		<tr>
			<td>Single channel pipette</td>
			<td>Rainin</td>
			<td>17008652</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus salivarius genomic DNA</td>
			<td>American Type Culture Collection</td>
			<td>BAA-1024D-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sweetened condensed milk</td>
			<td>California Farms</td>
			<td>B09TGQ7WV8</td>
			<td></td>
		</tr>
		<tr>
			<td>VSL#3</td>
			<td>VSL#3</td>
			<td>B07WX1LVHL</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glucuronidase from Helix pomatia</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>G7017</td>
			<td></td>
		</tr>
	</tbody>
</table>

use of micropipette-guided drug administration as an alternative method to oral gavage in rodent models

Oral gavage (OG) with the use of a cannula attached to a syringe is one of the most common methods used to deliver precise dosing of compounds to the stomach of research animals. Unfortunately, this method comes with difficulties for both the operator and the research animal. Studies have shown that OG may lead to complications, including esophagitis, perforation of the esophagus, and inadvertent tracheal drug administration. In addition, OG is associated with increased plasma and fecal corticosterone levels (due to stress), altered blood pressure, and increased heart rate, which could negatively influence or bias study results. A previously developed alternative method termed micropipette-guided drug administration (MDA) incentivizes the animal to consume treatments readily in a minimally invasive manner. Herein, we present examples of the use of the MDA technique with treatments reconstituted in different vehicles and demonstrate effective delivery of the varied treatments to multiple different mouse strains. We further demonstrate that MDA is a technique that decreases the timing and invasiveness of drug administration and does not affect the gut microbiome composition as assessed by quantitative analysis of core gut microbial species. Overall, MDA may offer a less stressful and effective alternative to OG.

MDA can be used in the oral delivery of bacterial strains in mouse models. C57BL/6J mice were treated with antibiotics (cefoxitin in the drinking water) for 2 days to clear commensal microbial communities before starting the MDA training session. The sweetened condensed milk/water solution was administered once daily consecutively for 3 days prior to treatment administration. Mice were briefly restrained by gentle scruff during MDA treatment administration. On day 4, mice were treated once with PBS (nega...

Use of Micropipette-Guided Drug Administration as an Alternative Method to Oral Gavage in Rodent Models

Here, we describe micropipette-guided drug administration (MDA) as an alternative method to oral gavage that incentivizes the research animal to ingest treatments readily with minimal stress and discomfort.

Oral gavage (OG) with the use of a cannula attached to a syringe is one of the most common methods used to deliver precise dosing of compounds to the ...

use-micropipette-guided-drug-administration-as-an-alternative-method

Research

JoVE Journal

Cancer Research

703 Views.  Johns Hopkins University School of Medicine. Here, we describe micropipette-guided drug administration (MDA) as an alternative method to oral gavage that incentivizes the research animal to ingest treatments readily with minimal stress and discomfort. 

Video: Use of Micropipette-Guided Drug Administration as an Alternative Method to Oral Gavage in Rodent Models

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation.

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

Ovarian Cancer Patient-Derived Organoid Models for Pre-Clinical Drug Testing

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments is crucial to advancing healthcare and improving patient outcomes. Organoids are in-vitro three-dimensional multicellular miniature organs. Patient-derived organoid (PDO) models of ovarian cancer may be optimal for drug screening because they more accurately recapitulate tissues of interest than two-dimensional cell culture models and are inexpensive compared to patient-derived xenografts. In addition, ovarian cancer PDOs mimic the variable tumor microenvironment and genetic background typically observed in ovarian cancer. Here, a method is described that can be used to test conventional and novel drugs on PDOs derived from ovarian cancer tissue and ascites. A luminescence-based adenosine triphosphate (ATP) assay is used to measure viability, growth rate, and drug sensitivity. Drug screens in PDOs can be completed in 7-10 days, depending on the rate of organoid formation and drug treatments.

We present a protocol that can be used to conduct therapeutic drug testing with patient-derived ovarian cancer organoids.

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments ...

Automated Kinetic Imaging for Drug Assessment in Patient-Derived Tumor Organoids

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Author Spotlight: Developing Multiplexed Kinetic Assays for Organoid-Based Drug Response Analysis

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...

Optimized Venous Thrombosis Assay in a Mouse Cancer Model Using Vascular Clips

Venous Thrombosis Assay in a Mouse Model of Cancer

This methodology paper highlights the surgical nuances of a rodent model of venous thrombosis, specifically in the context of cancer-associated thrombosis (CAT). Deep venous thrombosis is a common complication in cancer survivors and can be potentially fatal. The current murine venous thrombosis models typically involve a complete or partial mechanical occlusion of the inferior vena cava (IVC) using a suture. This procedure induces a total or partial stasis of blood and endothelial damage, triggering thrombogenesis. The current models have limitations such as higher variability in clot weights, significant mortality rate, and prolonged learning curve. This report introduces surgical refinements using vascular clips to address some of these limitations. Using a syngeneic colon cancer xenograft mouse model, we employed customized vascular clips to ligate the infrarenal vena cava. These clips allow residual lip space similar to a 5-0 polypropylene suture after IVC ligations. Mice with the suture method served as controls. The vascular clip method resulted in a consistent reproducible partial vascular occlusion and greater clot weights with less variability than the suture method. The larger clot weights, greater clot mass, and clot to the IVC luminal surface area were expected due to the higher pressure profile of the vascular clips compared to a 6-0 polypropylene suture. The approach was validated by gray scale ultrasonography, which revealed consistently greater clot mass in the infrarenal vena cava with vascular clips compared to the suture method. These observations were further substantiated with the immunofluorescence staining. This study offers an improved method to generate a venous thrombosis model in mice, which can be employed to deepen the mechanistic understanding of CAT and in translational research such as drug discovery.

Author Spotlight: Advancing Cancer Associated Thrombosis Research in Rodent Models 

This article aims to present an optimized method for assessing venous thrombosis in a mouse cancer model, using vascular clips to achieve venous ligation. Optimization minimizes variability in thrombosis-related measurements and enhances relevance to human cancer-associated venous thrombosis.

This methodology paper highlights the surgical nuances of a rodent model of venous thrombosis, specifically in the context of cancer-associated thrombosis ...

Validation of FAM83A as a Target Gene in Cervical Cancer

Knockdown of FAM83A to Verify Its Role in Cervical Cancer Cell Growth and Cisplatin Sensitivity

The exploration of tumor target genes holds paramount importance for the prevention and treatment of cervical cancer. In this study, we outline the steps involved in the identification of a tumor target gene FAM83A in cervical cancer. First, the Cancer Genome Atlas dataset was employed to validate the expression and prognostic significance of FAM83A in women. A small interfering RNA (siRNA) was used for knockdown of the FAM83A gene in HeLa and C33a cells. Next, 5-ethynyl-2&#39;-deoxyuridine (EdU) staining was conducted to determine the effects on the proliferation capabilities of the tumor cells. Wound healing and porous membrane insert assays were performed to evaluate tumor cell migration and invasion abilities.
Western blotting was used to quantify apoptosis-related protein levels. JC-1 staining was employed to evaluate mitochondrial function alterations. Furthermore, cisplatin (diaminedichloroplatinum, DDP) intervention was used to assess the therapeutic potential of the target gene. Flow cytometry and colony formation assays were conducted to further validate the anticancer characteristics of the gene. As a result, FAM83A knockdown was shown to inhibit the proliferation, migration, and invasion of cervical cancer cells and sensitize these cells to cisplatin. These comprehensive methodologies collectively validate FAM83A as a tumor-associated target gene, holding promise as a potential therapeutic target in the prevention and treatment of cervical cancer.

Author Spotlight: Exploring the Role of FAM83A in Cervical Cancer 

Here, we show the procedures for FAM83A knockdown; the assays to detect its effects on proliferation, migration, and invasion of cervical cancer cells; and the sensitization of these cells to cisplatin. This study provides a promising target gene for cervical cancer and a reference for further drug research.

The exploration of tumor target genes holds paramount importance for the prevention and treatment of cervical cancer. In this study, we outline the steps ...