Name: generating and imaging mouse and human epithelial organoids from normal and tumor mammary tissue without passaging
Uploaded: 2022-11-11T14:30:00
Description: Watch this Scientific Journal Video about Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging at JoVE.com

This study was supported by funding provided by METAvivor, the Peter Carlson Trust, Theresa&#39;s Research Foundation, and the NCI/UTSW Simmons Cancer Center P30 CA142543. We acknowledge the assistance of the University of Texas Southwestern Tissue Management Shared Resource, a shared resource at the Simmons Comprehensive Cancer Center, which is supported in part by the National Cancer Institute under award number P30 CA142543. Special thanks to all members of the Chan Lab.

All mouse tissue utilized in this manuscript has been ethically collected in accordance with the Institutional Animal Care and Use Committee (IACUC) regulations and guidelines of the University of Texas Southwestern Medical Center. Likewise, all the patients consented prior to tissue donation under the oversight of an Institutional Review Board (IRB), and the samples were deidentified.
NOTE: This protocol describes the generation of organoids from primary tissue.
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Different methods have been described in the literature to generate tumor organoids. This protocol highlights a method for generating tumor organoids directly from the tumor without passaging. Using this method, tumor organoids are producible within hours of initiating the procedure and generate close to 100% viable organoids compared to 70% reported in the literature31. In comparison, other methods require serial passaging of cells into organoids over several weeks. Thus, the downstream applicati...

The ability to model epithelial cells in vitro has been the foundation of modern biomedical research because it captures cellular features that are not accessible in vivo. For instance, growing epithelial cell lines in a two-dimensional plane can provide an assessment of the molecular changes that occur in an epithelial cell during proliferation1. Furthermore, measuring the dynamic regulation between signaling and gene expression is limited in in vivo systems2. In cancer research, cancer epithelial cell line modeling has enabled the identification of molecular drivers of disease progression and potential drug targets3. However, growing cancer epithelial cell lines on a two-dimensional plane has limitations, as most are genetically immortalized and modified, often clonal in nature, selected for their ability to grow in non-physiologic conditions, limited in their assessment of three-dimensional (3D) tumor tissue architecture, and do not adequately model microenvironment interactions within a realistic tissue environment4. These constraints are particularly evident in modeling metastasis, which in vivo includes several distinct biological stages, including invasion, dissemination, circulation, and colonization at the distant organ site5.
Cancer epithelial organoids have been developed to better recapitulate the 3D environment and behavior of tumors6,7,8. Organoids were first developed from single LRG5+ intestinal crypt cells and differentiated to represent the 3D structure of crypt-villus units that maintained the hierarchical structure of the small intestine in vitro9. This approach permitted real-time visualization and characterization of self-organizing tissue architecture under homeostatic and stress conditions. As a natural extension, cancer epithelial organoids were developed to model many different cancer types, including colorectal10, pancreatic11, breast12, liver13, lung14, brain15, and gastric cancers16. Cancer epithelial organoids have been exploited to characterize cancer evolution17,18 and metastatic spatiotemporal behaviors19,20 and interrogate tumor heterogeneity21, and test chemotherapies22. Cancer epithelial organoids have also been isolated and collected during ongoing clinical trials to predict patient response to anticancer agents and radiation therapy ex vivo8,23,24,25. Furthermore, systems incorporating cancer epithelial organoids can be combined with other non-cancer cells, such as immune cells, to form a more comprehensive model of the tumor microenvironment to visualize interactions in real-time, uncover how cancer epithelial cells change the fundamental nature of cytotoxic effector immune cells such as natural killer cells, and test potential immunotherapies and antibody-drug dependent cytotoxic activity26,27,28. This article demonstrates a method of generating epithelial organoids without passaging and embedding in collagen and basement extracellular matrix (ECM). Additionally, techniques for downstream imaging of isolated organoids are also shared.

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			<td>10 mM HEPES Buffer</td>
			<td>Gibco&#160;</td>
			<td>15630080</td>
			<td></td>
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			<td>100x Antibiotic-Antimycotic&#160;</td>
			<td>Gibco&#160;</td>
			<td>15240-096</td>
			<td></td>
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			<td>100x Glutamax</td>
			<td>Life Technologies&#160;</td>
			<td>35050-061</td>
			<td>Glutamine supplement</td>
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			<td>100x Insulin-Transferrin-Selenium (ITS)&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>51500-056</td>
			<td></td>
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			<td>100x Penicillin/Streptomycin (Pen/Strep)</td>
			<td>Sigma&#160;</td>
			<td>P4333</td>
			<td></td>
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			<td>10x DMEM</td>
			<td>Sigma&#160;</td>
			<td>D2429</td>
			<td></td>
		</tr>
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			<td>50 mL/0.2 &#181;m filter flask</td>
			<td>Fisher&#160;</td>
			<td>#564-0020</td>
			<td></td>
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			<td>Amphotericin B</td>
			<td>Life Technologies&#160;</td>
			<td>15290-018</td>
			<td></td>
		</tr>
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			<td>bFGF</td>
			<td>Sigma</td>
			<td>F0291</td>
			<td></td>
		</tr>
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			<td>BSA Solution (32%)</td>
			<td>Sigma&#160;</td>
			<td>#A9576</td>
			<td></td>
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			<td>Cholera Toxin&#160;</td>
			<td>Sigma&#160;</td>
			<td>C8052</td>
			<td></td>
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			<td>CO2-Independent Medium&#160;</td>
			<td>Gibco</td>
			<td>18045-088</td>
			<td></td>
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			<td>Collagenase A&#160;</td>
			<td>Sigma&#160;</td>
			<td>C2139</td>
			<td></td>
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			<td>Deoxyribonuclease I from bovine pancreas (DNase)</td>
			<td>Sigma</td>
			<td>D4263</td>
			<td></td>
		</tr>
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			<td>DMEM with 4500 mg/L glucose, sodium pyruvate, and sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture</td>
			<td>Sigma</td>
			<td>D6546</td>
			<td>Common basal medium</td>
		</tr>
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			<td>D-MEM/F12&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#10565-018</td>
			<td>Basal cell medium</td>
		</tr>
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			<td>Dulbecco&#39;s Phosphate Buffered Saline (D-PBS)&#160;</td>
			<td>Sigma</td>
			<td>#D8662</td>
			<td>PBS</td>
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			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma&#160;</td>
			<td>#F0926</td>
			<td></td>
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			<td>Gentamicin&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#15750-060</td>
			<td></td>
		</tr>
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			<td>Human epidermal growth factor (EGF)</td>
			<td>Sigma&#160;</td>
			<td>E9644</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone&#160;</td>
			<td>Sigma&#160;</td>
			<td>H0396</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin&#160;</td>
			<td>Sigma&#160;</td>
			<td>#I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel&#160;</td>
			<td>Corning&#160;</td>
			<td>#354230</td>
			<td>Basement Extracellular Matrix (BECM)</td>
		</tr>
		<tr>
			<td>NaOH (1 N)</td>
			<td>Sigma&#160;</td>
			<td>S2770</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Tail Collagen I</td>
			<td>Corning&#160;</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 media</td>
			<td>Fisher&#160;</td>
			<td>SH3002701</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>27250-018</td>
			<td></td>
		</tr>
	</tbody>
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generating and imaging mouse and human epithelial organoids from normal and tumor mammary tissue without passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

The images featured in Figure 1 provide an example of wild-type and tumorous mammary epithelial organoids from human and mouse tissues. An at-a-glance illustration of the method for isolating epithelial organoids through differential centrifugation is provided in the cartoon workflow in Figure 1A, showing that primary tissues from different species can be processed in near-identical ways while yielding epithelial tissue as shown in the brightfield images (<stron...

Watch this Scientific Journal Video about Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging at JoVE.com

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

This protocol is significant for the production of memory epithelial organoids that are generated without passaging. The main advantage of this technique is that organoids can be isolated from human and mouse breast and memory tissue after enzymatic digestion. We perform a series of differential centrifugation steps that isolate epithelial organoids without having to passage cells.An individual performing this technique may face challenges when embedding the tissues in collagen or matrix and plating them within a 96-well plate. Furthermore, using properly polymerized collagen and plating stable domes is the key to seeing invasion. To begin, collect the mammary tissue of a euthanized mouse by splaying mouse limbs and using four 19 gauge needles, pin the mouse by the paws to a board covered with an absorbent pad with the ventral side facing upward.Spray with 70%ethanol to smooth the fur down and sanitize the skin and wipe away feces with a gauze pad or tissue. Starting just above the anogenital region, cut upward from the midline using surgical scissors taking care not to pierce through the peritoneum. Upon reaching the chin, make lateral cuts down both clavicles and down the hind legs and pin the skin of the mouse taut to the board to expose mammary fat pads.After locating the inguinal and thoracic mammary fat pads on wild type mice, use forceps to elevate the mammary fat pad. Using the blunt end of sharp blunt scissors, create a pocket underneath the mammary fat pad away from the skin and cut away the mammary fat pad in one complete piece. Once mammary fat pads have been removed, rinse in PBS before placing them in a sterile tissue culture dish and then quickly transfer to a tissue culture hood.Mince the mammary tumors with a number 10 or number 11 scalpel to loosen tissue until it reaches a paste-like consistency. Transfer the minced tissue into a conical tube containing 10 to 30 milliliters of collagenase solution using a scalpel. To ensure all tissue is collected, pipette one milliliter of collagenase solution onto the tissue culture plate and back into the conical tube.Place the conical tube into a benchtop shaking incubator until the tissue becomes stringy and the collagenase solution becomes cloudy. Spin down 50 milliliters of solution for five to 10 minutes at 1, 500 RPM. Aspirate supernatant, and add 12 milliliters of the basal medium and mix by pipetting up and down three to four times or gently rotate the wrist while holding the tube 15 times.Again, spin down aspirate supernatant, add basal medium, and mix by pipetting or tube rotation as demonstrated earlier. Once the heaviest tissue fragments settle to the bottom collect the supernatant with a serological pipette and transfer it to a 15 milliliter conical tube. After each centrifugation, ensure that the pellet becomes increasingly opaque.Aliquot appropriate suspension of organoids into microcentrifuge tubes according to the organoid density relative to the amount of BECM per well. Centrifuge the microcentrifuge tubes for 10 minutes at 300 G at room temperature and discard the supernatant from the tubes. Move the tubes with organoid pellets to ice and add the appropriate volume of BECM to each microcentrifuge tube.Gently pipette up and down to resuspend the organoids in BECM, taking care not to produce bubbles. Slowly and carefully pipette the BECM suspended organoids onto the plating surface and fill all the empty wells with PBS to maintain humidity. Place the plate into a 37 degrees Celsius incubator for one hour to allow BECM to solidify and then cover with the appropriate volume of media.To prepare the collagen solution, combine 375 microliters of 10 times the concentration of DMEM, 100 microliters of one normal sodium hydroxide, and three milliliters of rat tail collagen I solution in a 15 milliliter conical tube. Pipette mix, taking care not to make bubbles, and titrate the solution to a pH of 7.2 to 7.4 with a small amount of sodium hydroxide or 10 times the concentration of DMEM as needed. Coat the bottom of the wells with the minimum amount of collagen required to fully cover the bottom of the well by placing a small amount of collagen and rock the plate from side to side to coat.Allow the collagen underlays to set at 37 degrees Celsius for 30 minutes to two hours. Aliquot the appropriate suspension of organoids into microcentrifuge tubes according to the organoid density relative to the amount of collagen per well. Store the collagen solution at four degrees Celsius and monitor polymerization by checking for fiber formation under a microscope every 10 minutes.Centrifuge the microcentrifuge tubes at 300 G for 10 minutes at room temperature and discard the supernatant from the tubes. Move the organoid pellets to ice and add the appropriate volume of collagen to each microcentrifuge tube. Gently pipette up and down to resuspend organoids in collagen, taking care not to produce bubbles.Slowly and carefully pipette the collagen suspended organoids onto the plating surface. The immunofluorescent images of embedded organoids in either basement extracellular matrix or collagen were obtained to study the inter-species tissue composition similarities. Organoids embedded in collagen matrix can be used for an invasion assay and analyzed by tracking the expansion of tendrils branching out from the organoid itself either through membrane tomato labeling or phalloidin staining of actin.Lastly, hematoxylin and eosin staining of paraffin-embedded organoids show that organoids maintain the same histology of breast cancer. It is crucial to keep BECM and collagen cold while pipetting gelled domes to avoid premature polymerization. Additionally, prolonged fixing of domes in PFA will lead to gelled solution if performing immunofluorescent staining.Organoids can also be used in in vitro and in vivo procedures such as drug screens and mouse models of metastasis. These methods can provide insights into therapeutic sensitivity of tumor tissue, metastatic properties, and immune interactions.

generating-imaging-mouse-human-epithelial-organoids-from-normal-tumor

Research

JoVE Journal

Cancer Research

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

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

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

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

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

This protocol provides detailed experimental steps to establish a three-dimensional in vitro culture of bladder tumor organoids derived from carcinogen-induced murine bladder cancer. Culture methods including passaging, genetic engineering, and orthotopic transplantation of tumor organoids are described.

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of mucin-secreting tumor cells in the peritoneal cavity. PMP can arise from various types of cancers, including appendiceal, ovarian, and colorectal, though appendiceal neoplasms are by far the most common etiology. PMP is challenging to study due to its (1) rarity, (2) limited murine models, and (3) mucinous, acellular histology. The method presented here allows real-time visualization and interrogation of these tumor types using patient-derived ex vivo organotypic slices in a preparation where the tumor microenvironment (TME) remains intact. In this protocol, we first describe the preparation of tumor slices using a vibratome and subsequent long-term culture. Second, we describe confocal imaging of tumor slices and how to monitor functional readouts of viability, calcium imaging, and local proliferation. In short, slices are loaded with imaging dyes and are placed in an imaging chamber that can be mounted onto a confocal microscope. Time-lapse videos and confocal images are used to assess the initial viability and cellular functionality. This procedure also explores translational cellular movement, and paracrine signaling interactions in the TME. Lastly, we describe a dissociation protocol for tumor slices to be used for flow cytometry analysis. Quantitative flow cytometry analysis can be used for bench-to-bedside therapeutic testing to determine changes occurring within the immune landscape and epithelial cell content.

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

We describe a protocol for the production, culture, and visualization of human cancers, which have metastasized to the peritoneal surfaces. Resected tumor specimens are cut using a vibratome and cultured on permeable inserts for increased oxygenation and viability, followed by imaging and downstream analyses using confocal microscopy and flow cytometry.

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of ...

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

Mouse mammary glands comprise ductal trees, which are lined by epithelial cells and have one opening at the tip of each nipple. The epithelial cells play a major role in mammary gland function and are the origin of most mammary tumors. Introducing genes of interest into mouse mammary epithelial cells is a critical step in evaluating gene function in epithelial cells and generating mouse mammary tumor models. This goal can be accomplished through the intraductal injection of a viral vector carrying the genes of interest into the mouse mammary ductal tree. The injected virus subsequently infects mammary epithelial cells, bringing in the genes of interest. The viral vector can be lentiviral, retroviral, adenoviral, or adenovirus-associated viral (AAV). This study demonstrates how a gene of interest is delivered into mammary epithelial cells through mouse mammary intraductal injection of a viral vector. A lentivirus carrying GFP is used to show stable expression of a delivered gene, and a retrovirus carrying Erbb2 (HER2/Neu) is used to demonstrate oncogene-induced atypical hyperplastic lesions and mammary tumors.

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

The present protocol describes intraductal injection of viral vectors via the teat to deliver genes of interest into the mammary epithelial cells.

Mouse mammary glands comprise ductal trees, which are lined by epithelial cells and have one opening at the tip of each nipple. The epithelial cells play ...

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Author Spotlight: Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

Fresh Primary Tumor Tissue Processing to Establish Tumor Organoids and Primary Fibroblasts

Begin by placing the tissue in a sterile tissue culture plate and removing excess media. Measure the tissue with a sterile metal ruler and photograph it. Add three milliliters of advanced DMEM/F-12 media to the cut tissue and pipette the tissue up and down to wash it.Then wash the tissue with three milliliters of PBS. Aspirate the medium from the tissue culture plate and cut it into small pieces using sterile blades and two milliliters of digestion media. Then collect the tissue into a 50-milliliter tube containing five milliliters total of the digestion medium.Incubate the tube at 37 degrees Celsius for one hour. Periodically mix the suspension by pipetting up and down. Inactivate the trypsin by adding five milliliters of advanced DMEM/F-12 to the tube.Then filter the sample through a 70-micrometer pore strainer to remove any large debris. Now pipette two milliliters of DMEM containing 10%FBS to the top of the filter, and use a pipette to collect the debris and tissue remnants for fibroblast culturing. Then plate the debris for fibroblast culture.Centrifuge the filtrate at room temperature at 200 to 300 G for five minutes, and then aspirate the supernatant. Add five milliliters of advanced DMF 12 to the pellet and centrifuge the suspension again at 300 G for five minutes. For red blood cell lysis, resuspend the pellet in four milliliters of ammonium chloride potassium lysis buffer, and transfer it to a 15-milliliter tube.Incubate the cells at room temperature for two minutes. After centrifuging the mixture at the same conditions as demonstrated previously, aspirate the supernatant. To the pellet, add five milliliters of advanced DMEM/F-12, and centrifuge again at four degrees Celsius.After aspirating the supernatant, add one milliliter of commercially available cell dissociation reagent to the pellet and incubate it for two to three minutes at room temperature. After incubation, centrifuge and aspirate the supernatant once again. Then resuspend the pellet in five milliliters of advanced DMEM/F-12.Dissociate cell clumps by pipetting the suspension multiple times with a P1000 pipette. Centrifuge the suspension and remove the supernatant. Next, obtain pre-chilled P1000 and P200 pipette tips, and use the cold tips fixed onto the P1000 pipette to resuspend the cell pellet in the membrane matrix, before placing the Falcon on ice to prevent solidification.Obtain the preheated plate from the incubator. Using the P200 pipette set at 48 microliters and using cold tips, create basement membrane matrix domes in the preheated plate. Then incubate it to solidify the basement membrane mix.Once the matrix is solidified, add two to two and a half milliliters of advanced DMEM/F-12, supplemented with the growth factors and inhibitors. Tumor cells were isolated and tumor organoids were established from fresh tissue over a 15-day period. Occasionally, large cell debris volumes prevent accurate visualization of developing tumor organoids.Developing organoids were observed to phenotypically vary from isolated, rounded to spheroid or aggregate-like cultures, depending on the tumor origin. Organoid cultures may be contaminated by fibroblasts, a common PI product of primary tumor cell culture. After approximately seven days of culture, the fibroblasts migrate from the basement membrane matrix domes and adhere to the cell plates, which may compromise optimal organoid growth.Fibroblast cultures are established from the tissue debris recovered from the filter. The cells migrate out of the tissue debris and adhere to the culture plates.