Name: isolation and functional assessment of human breast cancer stem cells from cell and tissue samples
Uploaded: 2020-10-02T14:00:00
Description: Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Vi takker medlemmerne af vores laboratorium for deres nyttige diskussioner og støtte. Vores forskning i brystkræftstamceller og tumormikromiljøet finansieres af tilskud fra Canadian Cancer Research Society Research Institute og U.S. Army Department of Defense Breast Cancer Program (Grant # BC160912). V.B. støttes af et Western Postdoctoral Fellowship (Western University), og både A.L.A. og V.B. støttes af Breast Cancer Society of Canada. CL understøttes af et Vanier Canada Graduate Scholarship fra Canadas regering.

Indsamling af patientafledte kirurgiske eller biopsiprøver direkte fra samtykkende brystkræftpatienter blev udført under godkendt human etisk protokol godkendt af det institutionelle etiske råd. Alle mus, der blev brugt til at generere patientafledte xenograftmodeller, blev vedligeholdt og anbragt i en institutionsgodkendt dyrefacilitet. Tumorvævet fra patientafledte xenograftmodeller ved hjælp af mus blev genereret i henhold til godkendt etisk protokol godkendt af den institutionelle dyreplejekomité.
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Brystkræftmetastase og resistens over for terapi er blevet en væsentlig årsag til dødelighed hos kvinder over hele verden. Eksistensen af en delpopulation af brystkræftstamceller (BCSC'er) bidrager til forbedret metastase 26,43,44,45,46 og terapiresistens21,47,48.</sup...

Forståelse af de cellulære og molekylære mekanismer i menneskelige brystkræftstamceller (BCSC'er) er afgørende for at tackle de udfordringer, der opstår i brystkræftbehandling. Fremkomsten af BCSC-konceptet går tilbage til begyndelsen af det 21. århundrede, hvor en lille population af CD44 + CD24- / lave brystkræftceller viste sig at være i stand til at generere heterogene tumorer hos mus 1,2. Efterfølgende blev det observeret, at humane brystkræftceller med høj enzymatisk aktivitet af aldehyddehydrogenase (ALDHhøj) også udviste lignende stamcellelignende egenskaber3. Disse BCSC'er repræsenterer en lille population af celler, der er i stand til selvfornyelse og differentiering, hvilket bidrager til den heterogene karakter af bulktumorer 1,2,3. Akkumulerende beviser tyder på, at ændringer i evolutionært bevarede signalveje driver BCSC-overlevelse og vedligeholdelse 4,5,6,7,8,9,10,11,12,13,14 . Derudover har det celleydre mikromiljø vist sig at spille en afgørende rolle i dikteringen af forskellige BCSC-funktioner15,16,17. Disse molekylære veje og de eksterne faktorer, der regulerer BCSC-funktionen, bidrager til tilbagefald af brystkræft, metastase18 og udvikling af resistens over for terapier 19,20,21, hvor den resterende eksistens af BCSC'er efter behandling udgør en stor udfordring for brystkræftpatienters samlede overlevelse22,23 . Præklinisk evaluering af disse faktorer er derfor meget vigtig for at identificere BCSC-målrettede terapier, der kan være gavnlige for at opnå bedre behandlingsresultater og forbedret samlet overlevelse hos brystkræftpatienter.
Flere in vitro human brystkræftcellelinjemodeller og in vivo human xenograft-modeller er blevet brugt til at karakterisere BCSC'er 24,25,26,27,28,29. Cellelinjernes evne til kontinuerligt at genbefolke efter hver efterfølgende passage gør disse til et ideelt modelsystem til at udføre omics-baserede og farmakogenomiske undersøgelser. Imidlertid undlader cellelinjer ofte at rekapitulere den heterogenitet, der observeres i patientprøver. Derfor er det vigtigt at supplere cellelinjedata med patientafledte prøver. Isolering af BCSC'er i deres reneste form er vigtig for at muliggøre detaljeret karakterisering af BCSC'er. Opnåelse af denne renhed afhænger af udvælgelsen af fænotypiske markører, der er specifikke for BCSC'er. I øjeblikket anvendes ALDHhøjCD44 + CD24-cellefænotype mest til at skelne og isolere humane BCSC'er fra bulk brystkræftcellepopulationer ved hjælp af fluorescensaktiveret cellesortering (FACS) for maksimal renhed 1, 3,26. Desuden kan egenskaberne ved isolerede BCSC'er såsom selvfornyelse, spredning og differentiering evalueres ved hjælp af in vitro- og in vivo-teknikker.
For eksempel kan in vitro-kolonidannende assays bruges til at vurdere en enkelt celles evne til selvfornyelse for at danne en koloni på 50 celler eller mere i nærvær af forskellige behandlingsbetingelser30. Mammosfæreassays kan også bruges til at vurdere selvfornyelsespotentialet for brystkræftceller under forankringsuafhængige forhold. Dette assay måler enkeltcellers evne til at generere og vokse som kugler (blanding af BCSC'er og ikke-BCSC'er) ved hver efterfølgende passage i serumfrie ikke-klæbende kulturbetingelser31. Derudover kan 3-dimensionelle (3D) kulturmodeller bruges til at vurdere BCSC-funktion, herunder celle-celle- og cellematrixinteraktioner, der nøje rekapitulerer in vivo-mikromiljøet og tillader undersøgelse af aktiviteten af potentielle BCSC-målrettede terapier32. På trods af de forskellige anvendelser af in vitro-modeller er det vanskeligt at modellere kompleksiteten af in vivo-forhold ved kun at bruge in vitro-assays. Denne udfordring kan overvindes ved hjælp af mus xenograft modeller til at evaluere BCSC adfærd in vivo. Sådanne modeller tjener især som et ideelt system til vurdering af brystkræftmetastase33, undersøgelse af interaktioner med mikromiljøet under sygdomsprogression 34, in vivo-billeddannelse 35 og til forudsigelse af patientspecifik toksicitet og effektivitet af antitumormidler 34.
Denne protokol giver en detaljeret beskrivelse af isoleringen af humane ALDH-høje CD44 + CD24- BCSC'er ved maksimal renhed fra bulkpopulationer af heterogene brystkræftceller. Vi giver også en detaljeret beskrivelse af tre in vitro-teknikker (kolonidannende assay, mammosfæreassay og 3D-kulturmodel) og et in vivo tumor xenograft-assay, der kan bruges til at vurdere forskellige funktioner af BCSC'er. Disse metoder vil være egnede til brug for forskere, der er interesserede i at isolere og karakterisere BCSC'er fra humane brystkræftcellelinjer eller primærpatientafledte brystkræftceller og tumorvæv med henblik på at forstå BCSC-biologi og / eller undersøge nye BCSC-målretningsterapier.

<table>
	<tbody>
		<tr>
			<td>7-Aminoactinomycin D (7AAD)</td>
			<td>BD</td>
			<td>51-68981E</td>
			<td>suggested: 0.25 &#181;g/1x106 cells</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher</td>
			<td>A18-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Aldehyde dehydrogenase (ALDH) substrate</td>
			<td>Stemcell Technologies</td>
			<td>1700</td>
			<td>Sold commerically as part of the ALDEFLOUR Assay kit; follow manufacturer&#39;s instructions for ALDH substrate preparation</td>
		</tr>
		<tr>
			<td>Basement membrane extract (BME)</td>
			<td>Corning</td>
			<td>354234</td>
			<td>Sold under the commercial name Matrigel</td>
		</tr>
		<tr>
			<td>Cell culture plates: 6 well</td>
			<td>Corning</td>
			<td>877218</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates: 60mm</td>
			<td>Corning</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates: 96-well ultra low attachment</td>
			<td>Corning</td>
			<td>3474</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer: 40 micron</td>
			<td>BD</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen</td>
			<td>Stemcell Technologies</td>
			<td>7001</td>
			<td>Prepare 1:30 dilution of 3 mg/mL collagen in PBS</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>11088807001</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Conical tubes: 50 mL</td>
			<td>Fisher scientific</td>
			<td>05-539-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal violet</td>
			<td>Sigma</td>
			<td>C6158</td>
			<td>Use 0.05% crystal violet solution in water for staining</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Stemcell Technologies</td>
			<td>7913</td>
			<td>5U/mL</td>
		</tr>
		<tr>
			<td>DMEM:F12</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td>1x, With L-glutamine and 15 mM HEPES</td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Sigma</td>
			<td>D5052</td>
			<td>0.1 mg/mL final concentration</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Avantor Seradigm Lifescience</td>
			<td>97068-085</td>
			<td>&#160;</td>
		</tr>
		<tr>
			<td>Flow tubes: 5ml</td>
			<td>BD</td>
			<td>352063</td>
			<td>Polypropylene round-bottom tubes</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>84124</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-Human CD24 antibody</td>
			<td>BD</td>
			<td>561646</td>
			<td>R-phycoerythrin and Cyanine dye conjugated Clone: ML5</td>
		</tr>
		<tr>
			<td>mouse anti-Human CD44 antibody</td>
			<td>BD</td>
			<td>555479</td>
			<td>R-phycoerythrin conjugated, Clone: G44-26</td>
		</tr>
		<tr>
			<td>N,N-diethylaminobenzaldehyde (DEAB)</td>
			<td>Stemcell Technologies</td>
			<td>1700</td>
			<td>Sold commerically as part of the ALDEFLOUR Assay kit; follow manufacturer&#39;s instructions DEAB preparation</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Wisent Inc</td>
			<td>311-425-CL</td>
			<td>1x, Without calcium and magnesium</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mammosphere Media Composition</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504-44</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Sigma</td>
			<td>F2006</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Bioshop</td>
			<td>ALB003</td>
			<td>04%</td>
		</tr>
		<tr>
			<td>DMEM:F12</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td>1x, With L-glutamine and 15 mM HEPES</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma</td>
			<td>E9644</td>
			<td>20 ng/mL</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>16634</td>
			<td>5 &#181;g/mL</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3D Organoid Media Composition</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A8301</td>
			<td>Tocris</td>
			<td>2939</td>
			<td>500 nM</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504-44</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>DMEM:F12</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td>1x, With L-glutamine and 15 mM HEPES</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma</td>
			<td>E9644</td>
			<td>5 ng/mL</td>
		</tr>
		<tr>
			<td>FGF10</td>
			<td>Peprotech</td>
			<td>100-26</td>
			<td>20 ng/mL</td>
		</tr>
		<tr>
			<td>FGF7</td>
			<td>Peprotech</td>
			<td>100-19</td>
			<td>5 ng/mL</td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>N-acetylcysteine</td>
			<td>Sigma</td>
			<td>A9165</td>
			<td>1.25 mM</td>
		</tr>
		<tr>
			<td>Neuregulin &#946;1</td>
			<td>Peprotech</td>
			<td>100-03</td>
			<td>5 nM</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma</td>
			<td>N0636</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>Peprotech</td>
			<td>120-10C</td>
			<td>100 ng/mL</td>
		</tr>
		<tr>
			<td>R-spondin3</td>
			<td>R&#38;D</td>
			<td>3500</td>
			<td>250 ng/mL</td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Sigma</td>
			<td>S7067</td>
			<td>500 nM</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>5 &#181;M</td>
		</tr>
	</tbody>
</table>

isolation and functional assessment of human breast cancer stem cells from cell and tissue samples

Brystkræftstamceller (BCSC'er) er kræftceller med arvelige eller erhvervede stamcellelignende egenskaber. På trods af deres lave frekvens er de store bidragydere til brystkræftinitiering, tilbagefald, metastase og terapiresistens. Det er bydende nødvendigt at forstå biologien af brystkræftstamceller for at identificere nye terapeutiske mål til behandling af brystkræft. Brystkræftstamceller isoleres og karakteriseres baseret på ekspression af unikke celleoverflademarkører såsom CD44, CD24 og enzymatisk aktivitet af aldehyddehydrogenase (ALDH). Disse ALDH-celler med høj CD44+CD24-celler udgør BCSC-populationen og kan isoleres ved fluorescensaktiveret cellesortering (FACS) til downstream-funktionelle undersøgelser. Afhængigt af det videnskabelige spørgsmål kan forskellige in vitro- og in vivo-metoder anvendes til at vurdere BCSC'ernes funktionelle egenskaber. Her leverer vi en detaljeret eksperimentel protokol til isolering af humane BCSC'er fra både heterogene populationer af brystkræftceller såvel som primært tumorvæv opnået fra brystkræftpatienter. Derudover fremhæver vi nedstrøms in vitro og in vivo funktionelle assays, herunder kolonidannende assays, mammosfæreassays, 3D-kulturmodeller og tumor xenograft-assays, der kan bruges til at vurdere BCSC-funktion.

Den beskrevne protokol tillader isolering af humane BCSC'er fra en heterogen population af brystkræftceller, enten fra cellelinjer eller fra dissocieret tumorvæv. For en given cellelinje eller vævsprøve er det afgørende at generere en ensartet enkeltcellesuspension for at isolere BCSC'er ved maksimal renhed, da forurenende ikke-BCSC-populationer kan resultere i variable cellulære reaktioner, især hvis undersøgelsens mål er at evaluere effektiviteten af terapeutiske midler rettet mod BCSC'er. Anvendelse af en str...

Denne eksperimentelle protokol beskriver isoleringen af BCSC'er fra brystkræftcelle- og vævsprøver samt in vitro - og in vivo-assays , der kan bruges til at vurdere BCSC-fænotype og funktion.

Isolering og funktionel vurdering af stamceller fra brystkræft fra celle- og vævsprøver

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

This technique will allow researchers to isolate breast cancer stem cells at maximum purity and viability for the performance of both in vitro and in vivo functional characterization assays. Isolated breast cancer stem cells can also be used to help elucidate the molecular events that control tumorigenicity and metastasis, and to design therapeutic strategies to target these cells. With minor modifications specific to the cell and tissue types of interest, this method can also be applied to other systems.To generate a single cell suspension from a cell culture, wash an 80%confluent breast cancer cell culture with PBS and treat the cells with an appropriate cell dissociation solution for five minutes at room temperature. Once the cells have detached, add five milliliters of culture medium to neutralize the cell dissociation solution and transfer the cells to a 50 milliliter conical tube for centrifugation. Resuspend the pellet in five milliliters of PBS for counting.After counting, centrifuge the cells again, then resuspend the cells at one times 10 to six cells per milliliter of ALDH substrate buffer. To generate a single cell suspension from a tissue sample, use surgical blades in a crisscross technique to mince the tumor tissue into approximately one millimeter size pieces. Place the pieces in a 50 milliliter conical tube containing 10 milliliters of dissociation buffer.Seal the tube with parafilm for a 40 minute incubation at 37 degrees Celsius in a shaker and sediment the digested tissue pieces by centrifugation. Resuspend the pellet in five milliliters of trypsin using a five milliliter pipette and incubate the tissue in a 37 degree water bath for five minutes. At the end of the incubation, vigorously pipette the tissue several times to release single cells and add enough DMEM/F-12 medium to bring the total volume in the tube to 25 milliliters.Collect the cells by centrifugation, resuspend the pellet in one milliliter of Displace DNAse, and incubate for five minutes in a 37 degree Celsius water bath. At the end of the incubation, bring the total volume to 10 milliliters with PBS. Mix the cells by pipetting and passage them through a 40 micron cell strainer into a 50 milliliter tube.After centrifugation, resuspend the cells in five milliliters of fresh PBS for counting. To perform a colony forming assay, after sorting, resuspend the cells in complete medium and add the appropriate number of breast cancer stem cells to each of the three tubes containing two milliliters of medium per tube, labeled one times 10 to the second, two times 10 to the second, or five times 10 to the second cells. Pipette up and down five times to thoroughly mix the cell suspension and plate the cells into individual wells of a six well plate.Gently swirl the plate to obtain uniform cell distributions and place the plate in the cell culture incubator until colonies appear. At the end of the incubation, wash the cells with one milliliter of PBS per well and stain the cells with 500 microliters of 0.05%crystal violet solution per well for 30 seconds. At the end of the incubation, wash the wells two times with two milliliters of water per well to remove the excess dye, and count and record the total number of colonies per well by light microscopy at a four or 10 times magnification.To perform a mammosphere assay, after sorting, resuspend the cells in complete mammosphere medium and plate the at a seating density of five times 10 of the second cells per square centimeter area per well in a 96 well ultra-low attachment cell culture plate. Before counting the number of mammospheres per well by light microscopy, place the plate in the cell culture incubator for five to 10 days. To set up a 3D culture model, carefully add 50 microliters of basement membrane extract to each well of a 96 well plate without bubbles and place the plate at 37 degrees Celsius for one hour to allow the solution to polymerize.Add 100 microliters of PBS after 10 minutes to avoid drying of the gel layer. At the end of the incubation, suspend the sorted cells to five to 50 times 10 to the third cells per 200 microliters of 3D culture medium concentration and replace the PBS in each well of the 96 well plate with 200 microliters of media containing cells. Then place the plate in the cell culture incubator for 10 to 14 days before assessing the cultures for organoid formation.Human breast cancer cells that exhibit enhanced ALDH enzymatic activity express high levels of CD44 and low to negative levels of expression of CD24, and can be classified as breast cancer stem cells. The proportion of breast cancer stem cells within the bulk population can vary between cell lines or patients and often depends on the disease stage, with more aggressive breast cancer typically displaying a higher proportion of breast cancer stem cells. The ability of a single breast cancer cell to self-renew and generate colonies of 50 cells can be assessed by colony forming assays.The ability of breast cancer stem cells to self-renew under anchorage-independent experimental conditions can be assessed by mammosphere assays. Culturing breast cancer cells and basement membrane extract allows breast cancer stem cells to form 3D structures that recapitulate in vivo conditions. In addition, in vivo mouse xenograft models can be used to understand the differences in growth, self-renewal, differentiation, and/or the tumor initiating ability of breast cancer stem cells compared to non-breast cancer stem cells or bulk cell populations.Following this procedure, the isolated breast cancer stem cells can be used in transcriptomic, kinomic, and metabolomic studies. This method provides researchers with an opportunity to explore the role of breast cancer stem cells in tumor initiation, metastasis, recurrence, and therapy resistance.

Research

JoVE Journal

Cancer Research

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

Etablering af Cancer Stem Cell Cultures fra Menneskelig Konventionel Osteosarkom

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

Ved hjælp af Functional Genomics Screening at identificere potentielt nyt lægemiddel mod cancer Cell sfæroide Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NF&#954;B Activity and Breast Cancer Stem Cells

Syntese og karakterisering af en Aspirin-fumarat prodrug, Hæmmer NFKB Aktivitet og brystkræft Stamceller

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an ...

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

Invasiv Opførsel af humane brystcancerceller i embryonale zebrafisk

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Mesenkymale stamceller isoleret fra Pulp væv og fælles kultur med kræftceller til at studere deres interaktioner

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

Anvendelse af menneskeskabte pluripotente stamceller til generering af tumor antigen-specifikke T-celler

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Opnåelse kræft stamceller kugler fra gynækologiske og brystkræft tumorer

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Isolering af Stem-lignende celler fra 3-dimensionelle sfæriske kulturer

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

Funktionel vurdering af BRCA1-varianter ved hjælp af CRISPR-medierede basiseditorer

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Modellering af brystkræft i humant brystvæv ved hjælp af et mikrofysiologisk system

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...