Name: isolation and functional assessment of human breast cancer stem cells from cell and tissue samples
Uploaded: 2020-10-02T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples at JoVE.com

We thank members of our laboratory for their helpful discussions and support. Our research on breast cancer stem cells and the tumor microenvironment is funded by grants from the Canadian Cancer Research Society Research Institute and the U.S. Army Department of Defense Breast Cancer Program (Grant # BC160912). V.B. is supported by a Western Postdoctoral Fellowship (Western University), and both A.L.A. and V.B. are supported by the Breast Cancer Society of Canada. C.L. is supported by a Vanier Canada Graduate Scholarship from the Government of Canada.

Collection of patient-derived surgical or biopsy samples directly from consenting breast cancer patients were carried out under approved human ethics protocol approved by the institutional ethic board. All mice used to generate patient-derived xenograft models were maintained and housed in an institution approved animal facility. The tumor tissue from patient-derived xenograft models using mice were generated as per approved ethics protocol approved by the institutional animal care committee.
1....

Breast cancer metastasis and resistance to therapy have become major cause of mortality in women worldwide. The existence of a sub-population of breast cancer stem cells (BCSCs) contributes to enhanced metastasis26,43,44,45,46 and therapy resistance21,47,48. Therefore...

Understanding the cellular and molecular mechanisms of human breast cancer stem cells (BCSCs) is crucial for addressing the challenges encountered in breast cancer treatment. The emergence of the BCSC concept dates back to the early 21st century, where a small population of CD44+CD24-/low breast cancer cells were found to be capable of generating heterogenous tumors in mice1,2. Subsequently, it was observed that human breast cancer cells with high enzymatic activity of aldehyde dehydrogenase (ALDHhigh) also displayed similar stem cell-like properties3. These BCSCs represent a small population of cells capable of self-renewal and differentiation, contributing to the heterogenous nature of bulk tumors1,2,3. Accumulating evidence suggest that alterations in evolutionarily conserved signaling pathways drive BCSC survival and maintenance4,5,6,7,8,9,10,11,12,13,14. In addition, the cell extrinsic microenvironment has been shown to play a pivotal role in dictating different BCSC functions15,16,17. These molecular pathways and the external factors regulating BCSC function contribute to breast cancer relapse, metastasis18 and development of resistance to therapies19,20,21, with the residual existence of BCSCs post-treatment posing a major challenge to the overall survival of breast cancer patients22,23. Pre-clinical evaluation of these factors is therefore very important for identifying BCSC-targeting therapies that could be beneficial for achieving better treatment outcomes and improved overall survival in breast cancer patients.
Several in vitro human breast cancer cell line models and in vivo human xenograft models have been used to characterize BCSCs24,25,26,27,28,29. The ability of cell lines to continuously repopulate after every successive passage makes these an ideal model system to perform omics-based and pharmacogenomic studies. However, cell lines often fail to recapitulate the heterogeneity observed in patient samples. Hence, it is important to complement cell line data with patient-derived samples. Isolation of BCSCs in their purest form is important for enabling detailed characterization of BCSCs. Achieving this purity depends on the selection of phenotypic markers that are specific to BCSCs. Currently, the ALDHhighCD44+CD24- cell phenotype is most commonly used to distinguish and isolate human BCSCs from bulk breast cancer cell populations using fluorescence activated cell sorting (FACS) for maximum purity1,3,26. Furthermore, the properties of isolated BCSCs such as self-renewal, proliferation, and differentiation can be evaluated using in vitro and in vivo techniques.
For example, in vitro colony forming assays can be used to assess the ability of a single cell to self-renew to form a colony of 50 cells or more in presence of different treatment conditions30. Mammosphere assays can also be used to assess the self-renewal potential of breast cancer cells under anchorage-independent conditions. This assay measures the ability of single cells to generate and grow as spheres (mixture of BCSCs and non-BCSCs) at each successive passage in serum-free non-adherent culture conditions31. Additionally, 3-Dimensional (3D) culture models can be used to assess BCSC function, including cell-cell and cell-matrix interactions that closely recapitulate the in vivo microenvironment and allow investigation of the activity of potential BCSC-targeted therapies32. Despite the diverse applications of in vitro models, it is difficult to model the complexity of in vivo conditions using only in vitro assays. This challenge can be overcome by use of mouse xenograft models to evaluate BCSC behavior in vivo. In particular, such models serve as an ideal system for assessing breast cancer metastasis33, investigating interactions with the microenvironment during disease progression34, in vivo imaging35, and for predicting patient-specific toxicity and efficacy of antitumor agents34.
This protocol provides a detailed description for the isolation of human ALDHhighCD44+CD24- BCSCs at maximum purity from bulk populations of heterogenous breast cancer cells. We also provide a detailed description of three in vitro techniques (colony forming assay, mammosphere assay, and 3D culture model) and an in vivo tumor xenograft assay that can be used to assess different functions of BCSCs. These methods would be appropriate for use by investigators interested in isolating and characterizing BCSCs from human breast cancer cell lines or primary-patient derived breast cancer cells and tumor tissue for the purposes of understanding BCSC biology and/or investigating novel BCSC-targeting therapies.

<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

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

The described protocol allows isolation of human BCSCs from a heterogenous population of breast cancer cells, either from cell lines or from dissociated tumor tissue. For any given cell line or tissue sample, it is crucial to generate a uniform single cell suspension to isolate BCSCs at maximum purity as contaminating non-BCSC populations could result in variable cellular responses, especially if the study aim is to evaluate the efficacy of therapeutic agents targeting BCSCs. Application of a stringent sorting strategy w...

Watch this Scientific Journal Video about Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples at JoVE.com

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

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

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.

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Cancer Research

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed &#34;cellular spheres&#34;. Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

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

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

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

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an anti-inflammatory drug to standard cancer regiments may improve patient outcome. One such drug, aspirin (acetylsalicylic acid, ASA), has been explored for cancer chemoprevention and anti-tumor activity. Besides inhibiting the cyclooxygenase 2-prostaglandin axis, ASA&#39;s anti-cancer activities have also been attributed to nuclear factor &#312;B (NF&#312;B) inhibition. Because prolonged ASA use may cause gastrointestinal toxicity, a prodrug strategy has been implemented successfully. In this prodrug design the carboxylic acid of ASA is masked and additional pharmacophores are incorporated.
This protocol describes how we synthesized an aspirin-fumarate prodrug, GTCpFE, and characterized its inhibition of the NF&#312;B pathway in breast cancer cells and attenuation of the cancer stem-like properties, an important NF&#312;B-dependent phenotype. GTCpFE effectively inhibits the NF&#312;B pathway in breast cancer cell lines whereas ASA lacks any inhibitory activity, indicating that adding fumarate to ASA structure significantly contributes to its activity. In addition, GTCpFE shows significant anti-cancer stem cell activity by blocking mammosphere formation and attenuating the cancer stem cell associated CD44+CD24- immunophenotype. These results establish a viable strategy to develop improved anti-inflammatory drugs for chemoprevention and cancer therapy.

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

This procedure will demonstrate how we synthesized and characterized the anti-NF&#954;B and anti-cancer stem cell activity of an aspirin-fumarate prodrug.

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

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 cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

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 ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

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

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

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

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

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

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

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

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C&#62;T, c.154C&#62;T, c.3847C&#62;T, c.5056C&#62;T, and c.4986+5G&#62;A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

People with BRCA1 mutations have a higher risk of developing cancer, which warrants accurate evaluation of the function of BRCA1 variants. Herein, we described a protocol for functional assessment of BRCA1 variants using CRISPR-mediated cytosine base editors that enable targeted C:G to T:A conversion in living cells.

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

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 fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

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 ...