This study was supported by ITT (Istituto Toscano Tumori) Grant Proposal 2010.

<ol>
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The authors declare that they have no competing financial interest.

CSCs have several properties that permit the identification of this particular cellular subset in the tumor bulk. On the basis of these characteristics, such as acquired resistance to cytotoxic chemotherapy agents for the overexpression of ATP-binding cassette multidrug efflux transporters28, 32, 33, or for the upregulation of the expression of detoxification enzymes such as ALDH32, for the expression of a particular surface marker, such as CD133, CD44, CD34, CD90, and others30, 34, 35, 41, several different methods to isolate CSCs have been developed42-44. One of these techniques is the sphere formation assay, which is based on the capacity of CSCs to grow under non-adherent conditions.
The ability of tissue stem cells and CSCs to form spheres was first described in studies on the identification of neural stem cells by Reynolds et al.37. Subsequently, Gibbs et al.38 used these studies to begin isolating CSCs from solid tumors, in particular, from bone sarcomas. We have decided to use the sphere formation assay method illustrated by Gibbs et al. to isolate CSCs from OSA cell lines obtained from conventional OS biopsies. We adapted the original method to improve the results of this assay and to facilitate its reproducibility for other cancer cell lines. With reference to the establishment of the sphere formation assay, we verified that plating 40,000 cells/well is a good practice for maintaining cells in isolation at the beginning of the assay. This trick is very important to avoid the possibility that the spherical colonies originate from cellular aggregation and not from the particular and exclusive capacity of a single CSC to grow under non-adherent conditions and form a spherical colony. This ability is a particularly critical point of this assay.
We also certified that to obtain a good rate of sphere formation, it is sufficient to refresh aliquots of growth factors every 3 d&#160;and not every day as described in the original method. In this study, we also established and extensively described a good method for isolating the spherical colonies that formed when cultured under non-adherent conditions. This step is critical in this assay because it is very important to try to isolate as many of the spheres as possible that are formed in each well without damaging them. It is also important to isolate only the spheres and not the single cells, which could remain in suspension for the duration of the assay. To overcome these critical points, we have developed a particular isolation method, which, as shown above, gave good results for CSC isolation. Obviously, there is the possibility that not all the spheres that formed can be recovered, but the loss percentage is very low. Indeed, we also have the possibility to use a filter with 40 &#181;m pores to isolate spheres after they become large (formed approximately&#160;100 - 200 cells).
This isolation stops sphere formation but allows the single cells, part of the methylcellulose residue, and the smallest spheres to be filtered. This elimination is performed by thorough filtration as described in the protocol.
Moreover, the selection of the largest spherical colonies through the 40 &#181;m mesh with the consequent loss of the smallest spherical colonies allows one to select the CSCs with the highest capacity to form spherical colonies and with greater stemness. All of these modifications were performed to improve the assay and to help researchers studying CSCs to understand and reproduce the most critical step of the original method of the sphere formation assay.
Among the studies regarding in vitro methods for isolating CSCs, this study aimed to show how this adapted sphere formation assay could be a good method for isolating CSCs from OSA cell lines. The adaptations to the original method and the detailed isolation technique described improve its efficacy. In a short time, good number of CSCs can be obtained and used for several experiments. Therefore, it is possible to rapidly confirm the stem-like phenotypes and, in particular, to study the double stem-like phenotype that characterizes OS-CSCs. Thus, this modified assay could be a good technique for isolating CSCs and studying their biology. In the future, this method, with additional adaptations, may also be used to isolate CSCs from other finite cancer cell lines obtained by biopsies of rare solid tumors.
The possibility of isolating CSCs from rare solid tumors, such as OS, not only permits the improvement of studies about this particular cancer but also extends to studies of different types of cancer to develop better methods for their isolation and to future studies of the biology of this important cellular subset. Therefore, as we have done in this study, it is important to improve the methods of CSC isolation through the study of CSC biology, with the final goal of finding molecular targets and developing a very specific anticancer therapy directed against this particular cellular subset, which is probably responsible for the maintenance of the primary tumor, the development of its recurrence, and the origin of metastases in several organs. The study of CSC biology is also important for finding therapies that could be incisive in the cure of cancers, such as OS, for which the survival rate after neoadjuvant treatment remains very poor.

Sarcomas are a heterogeneous group of rare malignant connective tissue tumors originating predominantly from the embryonic mesoderm1. The different types include bone sarcomas and soft tissue sarcomas. Bone sarcomas, a group of relatively uncommon primary tumors, consist of several subtypes, including osteosarcoma (OS). OS, one of the most common primary tumors of the bone, is a mesenchymal malignancy that exhibits extensive clinical, histological, and molecular heterogeneities2, 3. Unfortunately, OS occurs predominantly in children and in young adults4, 5 and represents 60% of the common histologic subtypes of bone sarcoma in childhood6, 7. OS usually affects the skeletal areas, which are characterized by rapid bone growth (e.g., the metaphysis of long bones). Among the histologically different subtypes of OS, conventional OS, also called medullary or central OS, has a high grade of malignancy and a quota share of 80%8. This 80% is composed of 60% conventional osteoblastic OS, 10% chondroblastic OS, and 10% fibroblastic OS6, 8-10. Other OS subtypes include anaplastic, telangiectatic, giant cell-rich and small cell OS. Despite advances in combined surgery and chemotherapy in OS management, the outcome remains poor, with a long-term survival rate of 65-70% in patients without metastasis11, 12. Distant recurrences frequently occur as pulmonary metastases or, less frequently, as metastases to distant bones and local recurrences13. Metastases are often resistant to conventional treatments. This resistance is the reason why the 10-year disease-free survival is approximately 30% in patients with metastatic disease at diagnosis14, 15.
As with normal tissue, cancer tissue is composed of a heterogeneous collection of cell types. Cells within the tumor seem to correspond to different stages of development. Within any normal tissue resides a subpopulation of cells with the ability to selfrenew, thus providing progenitors and mature cells for tissue homeostasis. Similarly, cancer is composed of a similar heterogeneous population of cells at different stages of development, with different degrees of proliferation and tumorigenic potential. A subset of these cancer cells, termed Cancer stem Cells (CSCs), constitutes a reservoir of selfsustaining cells with the exclusive ability to selfrenew and maintain the malignant potential of tumors, thus generating the different cell lineages that constitute the tumor bulk16. In the 1990s, studies on acute myeloid leukaemia provided the first compelling evidence for the existence of CSC subpopulations17, 18. CSCs have since been isolated from a great number of solid tumors19, thereby becoming one of the most researched topics in cancer research. CSCs may indeed arise from normal stem cells by mutations in genes that make the normal stem cells cancerous20-23. Multiple transforming mutations and interactions with the microenvironment could also contribute to&#160;healthy progenitors and mature cells acquiring the selfrenewal capacity and immortality that typify CSCs. There are several hypotheses about this transformation. Healthy progenitors, healthy mature cells, and cancer cells, can dedifferentiate to stem cells, obtaining a stem-like phenotype by activating selfrenewal-associated genes24-28. Despite several recent studies, the origins of CSCs have yet to be discovered.
A particular characteristic of CSCs is that their capacity to resist the multi-therapy approach, which consists of combined surgery and chemotherapy with different drugs. Recent studies have shown that CSCs may also acquire resistance to cytotoxic chemotherapy agents. Possible explanations for this resistance include the overexpression of ATP-binding cassette (ABC) multidrug transporter (i.e.,&#160;MDR1 and BCRP1), overexpression of chemotherapy metabolizing enzymes such as aldehyde dehydrogenase 1 (ALDH1), and/or changes in cell cycle kinetics30-33. The direct consequence of all these concepts that have been described thus far is that a cancer therapy would be efficient only if the CSC subpopulation were completely eliminated, while local recurrence or distant metastasis could occur if even a single CSC survived.
The discovery of CSCs in human sarcomas34, particularly OS35, or in any other bone and soft tissue cancers, has great clinical importance because it offers a possible explanation as to why many treatments seem to be effective initially, but the patients later relapse. Therefore, the hope for the future battle against conventional OS is to find new and specific targeted therapies based on the development of innovative drugs directed at OS-CSCs&#160;thanks to the molecular characterization of this subpopulation and to the study of CSC biology.
In 1992, Reynolds and colleagues, who were investigating whether a subset of stem cells was present in the adult mammalian brain, developed a method to isolate cells suspected to be stem-like cells36, 37. This method is based on the particular ability of these cells to form spherical colonies when grown under nonadherent conditions. Similar techniques were employed by Gibbs and colleagues in 2005 to study a subpopulation of stem-like cells in bone sarcomas38. To isolate and characterize OS-CSCs from primary cell cultures of different types of conventional OS, we decided to adapt this technique for OS cell lines.
Here, we describe this adapted method of the sphere formation assay, termed &#34;sarcosphere assay&#34;, which can be used to isolate OS-CSCs from finite primary cell lines derived from human biopsies of conventional OS. We also describe all the techniques used to validate the stem-like CSC phenotype of the cell lines isolated by this assay: 1) evaluation of the expression of genes that characterize pluripotent Embryonic Stem Cells (ESCs) and of the CD133 gene, which is a marker of CSCs; 2) colony-forming unit (CFU) assay; 3) evaluation of the ability of these cells to differentiate into osteoblasts and adipocytes under appropriate differentiation conditions; 4) study of the surface markers of Mesenchymal Stem Cells (MSCs) (i.e.,&#160;CD44, CD105 and Stro-1) by immunofluorescence staining and by flow cytometric analysis; 5) evaluation of the ALDH activity of these cells.

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.

Watch this Scientific Journal Video about Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma at JoVE.com

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

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 ...

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Cancer Research

18.9K Views.  University of Florence. 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.

Video: Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better understand the molecular mechanisms underlying glioma cell migration, an ability to study the interaction between hGCs and axons within the tumor microenvironment is essential. In order to model this cellular interaction, we developed a mixed culture system consisting of hGCs and dorsal root ganglia (DRG) axon-oligodendrocyte co-cultures. DRG cultures were selected because they can be isolated efficiently and can form the long, extensive projections which are ideal for migration studies of this nature. Purified rat oligodendrocytes were then added on purified rat DRG axons and induced to myelinate. After confirming the formation of compact myelin, hGCs were finally added to the co-culture and their interactions with DRG axons and oligodendrocytes was monitored in real-time using time-lapse microscopy. Under these conditions, hGCs form tumor-like aggregate structures that express GFAP and Ki67, migrate along both myelinated and non-myelinated axonal tracks and interact with these axons through the formation of pseudopodia. Our ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of hGC migration and could potentially be used for in vitro drug efficacy testing.

Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

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 ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

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.

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 ...

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (&#947;&#948;) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure &#947;&#948; T cells drug product for first-in-human use of allogeneic &#947;&#948; T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. 
The lymphocyte-rich fraction is utilized, and the &#947;&#948; T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of &#947;&#948; T cells, a clinical-grade magnetic cell-separation device and TCR&#945;&#946; beads are used to deplete contaminating T-cell receptor (TCR)&#945;&#946; T cells. The highly enriched &#947;&#948; T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched &#947;&#948; T cells in co-culture with the aAPCs facilitates the manufacture of highly pure &#947;&#948; T cells with an average fold expansion of &gt;229,000-fold from healthy donor blood.

Expansion and Enrichment of Gamma-Delta &#947;&#948; T Cells from Apheresed Human Product

Presented is a protocol for the expansion of Gamma-Delta (&#947;&#948;) T cell drug product. Lymphocytes are isolated by elutriation and &#947;&#948;-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The &#947;&#948; cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded.

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...