Name: Författare Spotlight: Utforska strategier för framgångsrikt immunsvar mot tumörer
Uploaded: 2024-08-16T13:00:00
Description: Human tumor samples hold a plethora of information about their microenvironment and immune repertoire. Effective dissociation of human tissue samples into ...

Denna studie stöddes av Adelson Medical Research Foundation (AMRF), Melanoma Research Alliance (MRA) och U54CA224068. Figur 1, bild 4 och figur 5 skapades med BioRender.com.

Denna studie följde alla institutionella riktlinjer för mänsklig vävnadsprovtagning. Informerat samtycke erhölls från patienter och identifierbara provdata anonymiserades. Proverna samlas in i operationssalen, placeras i en lösning av RPMI, saltlösning eller PBS, och bekräftas vara cancerogena via patologiavdelningen innan de används i forskning. Alla steg, utom när det anges, bör utföras vid 4 °C eller på is. Arbeta inuti en biosäkerhetshuv när du bearbetar mänsklig vävnad. Se materialtabellen...

Tumördissociationsprotokoll för analys av encells-RNA-sekvensering

<ol>
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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=High-throughput+RNA+isoform+sequencing+using+programmed+cDNA+concatenation.">High-throughput RNA isoform sequencing using programmed cDNA concatenation.</a> Nat Biotechnol. 42 (4), 582-586 (2024).</li><li>Xing, Y., 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=The+effect+of+cell+isolation+methods+on+the+human+transcriptome+profiling+and+microbial+transcripts+of+peripheral+blood.">The effect of cell isolation methods on the human transcriptome profiling and microbial transcripts of peripheral blood.</a> Mol Biol Rep. 48 (4), 3059-3068 (2021).</li><li>Ma, F., Hernandez, G. E., Romay, M., Iruela-Arispe, M. 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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=Reliable+approaches+to+extract+high-integrity+RNA+from+skin+and+other+pertinent+tissues+used+in+pain+research.">Reliable approaches to extract high-integrity RNA from skin and other pertinent tissues used in pain research.</a> Pain Rep. 5 (2), e818 (2020).</li><li>Montanari, 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=Automated-mechanical+procedure+compared+to+gentle+enzymatic+tissue+dissociation+in+cell+function+studies.">Automated-mechanical procedure compared to gentle enzymatic tissue dissociation in cell function studies.</a> Biomolecules. 12 (5), 701 (2022).</li><li>Yosefov-Levi, O., Tornovsky, S., Parnas, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+tissue+dissection+to+isolate+viable+single+cells.">Pancreatic tissue dissection to isolate viable single cells.</a> J Vis Exp. 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Detta protokoll beskriver dissociation av humana eller murina tumörprover till en encellssuspension för scRNA-sekvensering med hjälp av 10x Genomics mikrofluidiksystem. Vid bearbetning av vävnader som ofta kommer från sällsynta cancerformer eller som ingår i pågående kliniska prövningar eller långa experiment för murina tumörer, måste provet bevaras optimalt och noggrant mellan alla steg. Alla steg bör utföras snabbt på is eller vid 4 °C för att behålla de ursprungliga cellulära RNA-profilerna och f?...

Även om många framsteg inom cancerforskningen har lett till utveckling och FDA-godkännande av läkemedel som riktar sig mot hämmande receptorer som uttrycks på immun- och tumörceller, så kallade checkpointblockadhämmare, är det fortfarande svårt att hitta behandlingsresistens och att identifiera mekanismer som driver patientrespons1. Den komplexa utmaningen att karakterisera tumörheterogenitet utifrån dess molekylära mångfald och det intrikata samspelet mellan tumörceller och immunmikromiljön kräver nya metoder för att dissekera detta komplexa ekosystem med encellsupplösning. Att förstå de molekylära komplexiteterna i tumörer och deras mikromiljöer är avgörande för att utveckla terapeutiska strategier och dechiffrera den komplexa biologin som ligger till grund för cancerprogression2. Single-cell RNA-sekvensering (scRNA-seq) har blivit allt mer populärt eftersom det ger högupplöst analys av enskilda celler i komplexa tumörprover 3,4.
Tumörheterogenitet, som omfattar genetiska, epigenetiska och fenotypiska diversiteter, utgör en utmaning när det gäller att avslöja komplexiteten i cancerbiologi5. De konventionella metoderna för bulk-RNA-sekvensering tenderar att dölja de unika uttrycksprofilerna och fenotyperna av heterogena cellpopulationer som finns i tumörer, eftersom dessa metoder beräknar medelvärdet av signaler över hela cellpopulationer4. Däremot tillåter scRNA-seq dissektion av enskilda celltyper, vilket avslöjar olika genuttryck, repressionsmönster och cellulära tillstånd som annars förbises 4,6. Förutsättningen för scRNA-seq ligger i dess förmåga att avkoda enskilda cellers genetiska och molekylära signaturer, vilket är oerhört lovande när det gäller att upptäcka nya cancerterapier7.
Genom att dechiffrera genuttrycksprofilerna hos tumörceller och de omgivande stroma- och immuncellerna kan forskare identifiera nya terapeutiska mål och utveckla precisionsstrategier för genetisk medicin som är skräddarsydda för enskilda patienter6. Dessutom ger dissekering av immunrepertoaren inom tumörmikromiljön viktiga insikter om samspelet mellan tumörceller och immuneffektorer, vilket banar väg för immunterapeutiska interventioner3. Trots framsteg när det gäller att använda scRNAseq på kliniska prover kan flera utmaningar under bearbetningsstegen skada cellens RNA-integritet, livskraft och kvaliteten på de data som genereras. Dessutom kan bearbetning av vävnad med låg cellviabilitet öka de omgivande RNA-nivåerna i de droppar som genereras under inkapslingen av enskilda celler, vilket resulterar i korskontaminering och felaktig annotering av celltyper, medan långa dissociationsprotokoll som utförs vid 37 °C kan leda till uppreglering av en gensignatur för stressrespons i flera celltyper 8,9, 10. veckor
Den stegvisa proceduren (Figur 1A) som beskrivs i detta protokoll börjar med snabb mekanisk och enzymatisk dissociation av tumörer, följt av filtrering och avlägsnande av döda celler, beroende på livskraften hos varje tumörprov. För närvarande har denna procedur verifierats i melanom11, kolorektal12, skivepitelcancer13 i huvud och hals, tumörer i bukspottkörteln och lungorna (opublicerade data). Dessa tekniker säkerställer generering av livskraftiga cellsuspensioner av hög kvalitet som är avgörande för nedströmsanalyser14. Noterbart är att det är absolut nödvändigt att ta bort röda blodkroppar, som saknar syntetiserat RNA, för att rena provet15. Efterföljande utvärdering av cellantal och eliminering av döda celler fungerar som en förutsättning för framgångsrik 10x Genomics Gel-bead-in Emulsion (GEM) generering, streckkodning och ökar återvinningen av transkript och sekvenserade celler utan att äventyra uteslutningen av känsliga populationer (t.ex. neutrofiler och epitelceller)16. Dessa steg är grundläggande för att låsa upp potentialen för analys av encellsupplösning i tumörprover 9,10, avslöja nya genuttrycksprofiler och immunsignaturer som är nödvändiga för att driva innovativa terapeutiska interventioner och identifiera nya tumörsårbarheter.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>1x PBS</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipette</td>
			<td>Corning</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L low-retention pipette tips</td>
			<td>Rainin</td>
			<td>30389213</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L low-retention wide pipette tips</td>
			<td>Rainin</td>
			<td>30389218</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L pipette tips</td>
			<td>Rainin</td>
			<td>30389212</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Magnetic Separator</td>
			<td>10x Genomics</td>
			<td>120250</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Corning</td>
			<td>430052</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL aspirating pipette</td>
			<td>Corning</td>
			<td>357558</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#956;L low-retention pipette tips</td>
			<td>Rainin</td>
			<td>30389226</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#956;L pipette tips</td>
			<td>Rainin</td>
			<td>30389225</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L low-retention pipette tips</td>
			<td>Rainin</td>
			<td>30389240</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L pipette tips</td>
			<td>Rainin</td>
			<td>30389239</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Polypropylene Conical Tube</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>60 x 15 mm Tissue Culture Dish</td>
			<td>Falcon</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>Gibco</td>
			<td>A10492-1</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 1 mL syringe</td>
			<td>Becton, Dickinson and Company</td>
			<td>3090659</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-Line Hemocytometer</td>
			<td>Hausser Scientific</td>
			<td>551660</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Solution</td>
			<td>Sigma Aldrich</td>
			<td>2115-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Ranger&#160;</td>
			<td>10x Genomics</td>
			<td>N/A</td>
			<td>https://www.10xgenomics.com/support/software/cell-ranger/latest</td>
		</tr>
		<tr>
			<td>Cell counting slides for TC20 cell counter</td>
			<td>Bio-Rad</td>
			<td>1450015</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrics 30&#956;m sterile disposable filters</td>
			<td>Sysmex</td>
			<td>04-004-2326</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrics 50&#956;m sterile disposable filters</td>
			<td>Sysmex</td>
			<td>04-004-2327</td>
			<td></td>
		</tr>
		<tr>
			<td>Dead Cell removal Kit&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-101</td>
			<td>Store at -20 &#176;C; kit 2</td>
		</tr>
		<tr>
			<td>Dissecting Forceps, Fine Tip</td>
			<td>VWR</td>
			<td>82027-386</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA LoBind Microcentrifuge tubes, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>22431021</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySepTM Dead Cell Removal 
			(Annexin V) Kit</td>
			<td>STEMCELL Technologies</td>
			<td>17899</td>
			<td>Store at 4 &#176;C; Kit 1</td>
		</tr>
		<tr>
			<td>Eppendorf centrifuge 5425R</td>
			<td>Eppendorf</td>
			<td>5406000640</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf centrifuge 5910R</td>
			<td>Eppendorf</td>
			<td>2231000657</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Easypet 3 Electronic Pipette controller</td>
			<td>Eppendorf</td>
			<td>EP4430000018</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Thermomixer F1.5 Model 5384</td>
			<td>Eppendorf</td>
			<td>EP5384000012</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td>Store at 4 &#176;C, use in a Biological hood</td>
		</tr>
		<tr>
			<td>German Stainless Scissors</td>
			<td>Fine Science Tools</td>
			<td>14568-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Dmi1 Microscope</td>
			<td>Leica Microsystems</td>
			<td>454793</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Multistand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-1000XLS+</td>
			<td>Rainin</td>
			<td>17014382</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-200XLS+</td>
			<td>Rainin</td>
			<td>17014391</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-20XLS+</td>
			<td>Rainin</td>
			<td>17014392</td>
			<td></td>
		</tr>
		<tr>
			<td>Quadro MACS Magnet</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-051</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Medium 1640 (1x)</td>
			<td>Gibco</td>
			<td>21870-076</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>TempAssure 0.2 mL PCR 8-Tube strips</td>
			<td>USA Scientific</td>
			<td>1402-4700</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue stain 0.4%</td>
			<td>Thermo Fisher Scientific</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Tumor Dissociation Kit, Human</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-929</td>
			<td>Store at -20 &#176;C, prepare enzymes according to kit instructions: 
			Reconstitute lyophilized Enzyme H vial in 3 mL of RPMI 1640 
			Reconstitute lyophilized Enzyme R vial in 2.7 mL of RPMI 1640 
			Reconstitute lyophilized Enzyme A vial in 1 mL of Buffer A supplied with the kit.</td>
		</tr>
		<tr>
			<td>Tumor Dissociation Kit, Mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-730</td>
			<td>Store at -20 &#176;C, prepare enzymes according to kit instructions: 
			Reconstitute lyophilized Enzyme D vial in 3 mL of RPMI 1640 
			Reconstitute lyophilized Enzyme R vial in 2.7 mL of RPMI 1640 
			Reconstitute lyophilized Enzyme A vial in 1 mL of Buffer A supplied with the kit.</td>
		</tr>
		<tr>
			<td>UltraPure Distilled Water</td>
			<td>Invitrogen</td>
			<td>10977-015</td>
			<td>Store at 4 &#176;C</td>
		</tr>
	</tbody>
</table>

dissociation of human and mouse tumor tissue samples for single-cell rna sequencing

Mänskliga tumörprover innehåller en uppsjö av information om deras mikromiljö och immunrepertoar. Effektiv dissociation av humana vävnadsprover till livskraftiga cellsuspensioner är en nödvändig input för pipelinen för encells-RNA-sekvensering (scRNAseq). Till skillnad från bulk-RNA-sekvenseringsmetoder gör scRNAseq det möjligt för oss att härleda den transkriptionella heterogeniteten i tumörprover på encellsnivå. Att införliva detta tillvägagångssätt under de senaste åren har lett till många upptäckter, såsom att identifiera immun- och tumörcellulära tillstånd och program associerade med kliniska svar på immunterapier och andra typer av behandlingar. Dessutom kan encellsteknik som tillämpas på dissocierade vävnader användas för att identifiera tillgängliga kromatinregioner T- och B-cellsreceptorrepertoar och uttrycket av proteiner med hjälp av DNA-streckkodade antikroppar (CITEseq).
Livskraften och kvaliteten på det dissocierade provet är kritiska variabler när man använder dessa tekniker, eftersom dessa dramatiskt kan påverka korskontamineringen av enskilda celler med omgivande RNA, kvaliteten på data och tolkning. Dessutom kan långa dissociationsprotokoll leda till eliminering av känsliga cellpopulationer och uppreglering av en gensignatur för stressrespons. För att övervinna dessa begränsningar utarbetade vi ett snabbt universellt dissociationsprotokoll, som har validerats på flera typer av mänskliga och murina tumörer. Processen börjar med mekanisk och enzymatisk dissociation, följt av filtrering, lys av rött blod och berikning av levande döda, lämpliga för prover med låg tillförsel av celler (t.ex. nålkärnbiopsier). Detta protokoll säkerställer en ren och livskraftig encellssuspension som är avgörande för en framgångsrik generering av Gel Bead-In Emulsioner (GEM), streckkodning och sekvensering.

Although many advancements in cancer research have led to the development and FDA approval of agents targeting inhibitory receptors expressed on immune and tumor cells, known as checkpoint blockade inhibitors, therapy resistance and identifying mechanisms that drive patient response remain challenging1. The complex challenge of characterizing tumor heterogeneity on its molecular diversity and the intricate interplay between tumor cells and the immune microenvironment necessitates new approaches to dissect this complex ecosystem at the single-cell resolution. Understanding the molecular intricacies within tumors and their microenvironments is pivotal for advancing therapeutic strategies and deciphering the complex biology underlying cancer progression2. Single-cell RNA sequencing (scRNA-seq) has become increasingly popular because it provides high-resolution analysis of individual cells within complex tumor samples3,4.
Tumor heterogeneity, encompassing genetic, epigenetic, and phenotypic diversities, poses a challenge in uncovering the intricacies of cancer biology5. The conventional methods of bulk RNA sequencing tend to obscure the unique expression profiles and phenotypes of heterogeneous cell populations present within tumors, as these methods average signals across entire cell populations4. In contrast, scRNA-seq allows the dissection of individual cell types, revealing diverse gene expression, repression patterns, and cellular states otherwise overlooked4,6. The premise of scRNA-seq lies in its capacity to decode individual cells&#39; genetic and molecular signatures, holding immense promise in discovering new cancer therapeutics7.
By deciphering the gene expression profiles of tumor cells and the surrounding stromal and immune cells, researchers can identify novel therapeutic targets and develop precision genetic medicine strategies tailored to individual patients6. Furthermore, dissecting the immune repertoire within the tumor microenvironment provides crucial insights into the interplay between tumor cells and immune effectors, paving the way for immunotherapeutic interventions3. Despite advances in using scRNAseq on clinical samples, several challenges during the processing steps can harm the cell&#39;s RNA integrity, viability, and quality of the data generated. Moreover, processing tissue with low cell viability can increase ambient RNA levels in the droplets generated during the encapsulation of individual cells, resulting in cross-contamination and incorrect annotation of cell types, while long dissociation protocols performed at 37 &#176;C can lead to the upregulation of a stress response gene signature in multiple cell types8,9,10.
The stepwise procedure (Figure 1A) outlined in this protocol commences with rapid mechanical and enzymatic dissociation of tumors, followed by filtration and the removal of dead cells, depending on the viability of each tumor sample. At present, this procedure has been verified in melanoma11, colorectal12, head and neck squamous cell carcinoma13, pancreatic and lung (unpublished data) tumor samples. These techniques ensure the generation of high-quality viable cell suspensions crucial for downstream analyses14. Notably, removing red blood cells, devoid of synthesized RNA, becomes imperative to purify the sample15. Subsequent evaluation of cell counts and the elimination of dead cells serve as a prerequisite for successful 10x Genomics Gel bead-in Emulsion (GEM) generation, barcoding, and increase the recovery of transcripts and sequenced cells without jeopardizing the exclusion of sensitive populations (e.g., neutrophils and epithelial cells)16. These steps are fundamental in unlocking the potential of single-cell resolution analysis within tumor samples9,10, uncovering novel gene expression profiles and immune signatures necessary for driving innovative therapeutic interventions, and identifying new tumor vulnerabilities.

Författare Spotlight: Utforska strategier för framgångsrikt immunsvar mot tumörer

Dissociation av vävnadsprover från humana tumörer och möss för encells-RNA-sekvensering

The scope of our research is to study the mechanisms of response or resistance in standard of care immunotherapies. Our lab is trying to understand what drives a successful immune response against tumors, and if we can identify these predictors of therapy to find targets that can overcome resistance. The biggest advancement in recent years has been the development and integration of single-cell RNA sequencing techniques.They enable us in real time to systematically study molecular features in individual cells in an unbiased manner, which is helpful in the dissection of these complex and evolving tumor ecosystems. While many preclinical models are being used to study the response mechanisms to immunotherapy, we still don't know the immunological determinants that are important in human immunity. Processing and analyzing patient samples in real time is crucial to our ability to investigate all cellular compartments within a given tumor.Using unbiased genomic and transcriptomic techniques, our lab discovered that defects in the antigen presentation machinery are associated with immunotherapy resistance in melanoma. More recently, using single-cell approaches, we discovered unique T cell states and myeloid cell polarities associated with patient outcomes in melanoma and head neck cancer, respectively. The biggest advantage of our protocol lies in its simplicity.It is a universal protocol adjusted for the dissociation of small and large tumor specimens from multiple types of cancers in a short period of time, ending in the generation of highly-viable, single-cell suspensions.

En melanombiopsi suspenderad i RPMI erhölls och lades omedelbart på is. Provet överfördes till ett 1,5 ml mikrocentrifugrör innehållande 420 μL RPMI och nedbrytningsenzymer, maldes i små bitar med hjälp av en sax och utsattes för efterföljande enzymatisk nedbrytning i 15 minuter i en 37 °C vertikalt placerad termisk mixer (figur 1). Efter nedbrytningen filtrerades provet genom ett 50 μm sterilt engångsfilter och filtret tvättades med färsk RPMI för att öka cellutbytet (<str...

Detta protokoll beskriver proceduren för att snabbt dissociera humana och mustumörprover för encells-RNA-sekvensering.

Human tumor samples hold a plethora of information about their microenvironment and immune repertoire. Effective dissociation of human tissue samples into ...

dissociation-human-mouse-tumor-tissue-samples-for-single-cell-rna

Research

JoVE Journal

Cancer Research

Dissociation of Human and Mouse Tumor Tissue Samples for Single-cell RNA Sequencing

Human tumor samples hold a plethora of information about their microenvironment and immune repertoire. Effective dissociation of human tissue samples into viable cell suspensions is a required input for the single-cell RNA sequencing (scRNAseq) pipeline. Unlike bulk RNA sequencing approaches, scRNAseq enables us to infer the transcriptional heterogeneity in tumor specimens at the single-cell level. Incorporating this approach in recent years has led to many discoveries, such as identifying immune and tumor cellular states and programs associated with clinical responses to immunotherapies and other types of treatments. Moreover, single-cell technologies applied to dissociated tissues can be used to identify accessible chromatin regions T and B cell receptor repertoire, and the expression of proteins, using DNA barcoded antibodies (CITEseq).
The viability and quality of the dissociated sample are critical variables when using these technologies, as these can dramatically affect the cross-contamination of single cells with ambient RNA, the quality of the data, and interpretation. Moreover, long dissociation protocols can lead to the elimination of sensitive cell populations and the upregulation of a stress response gene signature. To overcome these limitations, we devised a rapid universal dissociation protocol, which has been validated on multiple types of human and murine tumors. The process begins with mechanical and enzymatic dissociation, followed by filtration, red blood lysis, and live dead enrichment, suitable for samples with a low input of cells (e.g., needle core biopsies). This protocol ensures a clean and viable single-cell suspension paramount to the successful generation of Gel Bead-In Emulsions (GEMs), barcoding, and sequencing.

This protocol outlines the procedure for rapidly dissociating human and mouse tumor samples for single-cell RNA sequencing.

Rapid Dissociation of Tumor Tissue to Gain Enhanced Single-Cell Tumor Insights

To begin tumor digestion, pour the sample into a 60-millimeter tissue culture dish placed on ice. Pipette 420 microliters of media from the dish to a 1.5-milliliter microcentrifuge tube. Using tweezers, transfer the sample tissue to the tube containing media.Cut up the sample with clean scissors to pieces that are two to three millimeters in diameter. Add appropriate volumes of digestion enzymes and media to the sample. Vortex the tube for five seconds, and incubate it in a thermal mixer positioned vertically on its side.Place a 50-micrometer cell filter on top of a new 15-milliliter conical tube. And prime the filter with one milliliter of fresh RPMI media. Using a 1, 000-microliter lower tension wide pipette tip, load the sample to the filter and add the media.Then, with the back of a syringe plunger, grind and push the sample on the filter in a twisting motion. Wash the filter with five milliliters of media and any remaining media from the dish that contain the sample.

Snabb dissociation av tumörvävnad för att få förbättrade insikter om encelliga tumörer

Red Blood Cell Lysis in Digested Tumor Samples for Single-Cell RNA Sequencing

To begin, obtain the filtered and digested tumor sample. Centrifuge the sample at 450 G for five minutes at four degrees Celsius. Aspirate the media without disturbing the pellet and resuspend the pellet in ACK lysis buffer to remove red blood cells and record the volume used.Repeat the centrifugation and the lysis steps until the sample pellet has no visible red color after centrifugation. Then resuspend the sample pellet in fresh media. Mix 10 microliters each of trypan and blue and the dissociated cells in a 1.5 milliliter tube.Load 10 microliters on each side of a hemocytometer. to count the cells. Record the cell concentration.Total live cell count viability percentage, and final media volume after count.

Lys av röda blodkroppar i smälta tumörprover för encells-RNA-sekvensering

Dead Cell Removal from Dissociated Tumor for Single-Cell Sequencing

To begin, obtain the digested tumor tissue sample and lice the red blood cells. Centrifuge the sample at 450G for five minutes at four degrees Celsius. After removing the media, resuspend the pellet in one milliliter of the isolation media.After repeating the centrifugation, resuspend the pellet in 100 microliters of isolation media. Transfer 100 microliters of sample into a well of a 0.2 milliliter PCR strip tube. In a biosafety hood, add 10 microliters each of annexin V cocktail and biotin selection cocktail to the sample.Pipette mix the solution and incubate at room temperature for four minutes. Next, vortex the dextran-coated magnetic particles for 30 seconds. Mix 20 microliters of the beads and 60 microliters of isolation media with the sample.After three minutes, place the strip tube onto a 10x Genomics magnetic separator on the high setting for five minutes at room temperature. Then transfer the liquid from the strip tube without disturbing the beads into a new 1.5 milliliter low bind micro centrifuge tube. Centrifuge the tube as demonstrated earlier, and resuspend the pellet in an appropriate volume of our PMI media based on the pellet size.Finally, count the cells in a hemocytometer. Following dead cell removal, the live cell count was 4.9 times 10 to the power of 5 per milliliter, with viability above 90%