Name: <meta charset="utf8"></head><body>Auteur in de schijnwerpers: Validatie van reacties op kankertherapie met desmoplastische sferoïde modellen
Uploaded: 2024-09-27T13:00:00
Description: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of &#60;12%. The biggest barrier to therapy is the ...

<meta charset="utf8"></head><body>3D-PDAC-model dat desmoplasie vastlegt voor therapeutische evaluatie

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Axio Observer inverted microscope</td>
			<td>Carl Zeiss</td>
			<td>0450-354</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellometer Auto T4</td>
			<td>Nexcelom Bioscience LLC</td>
			<td>Auto-T4</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, powder, high glucose</td>
			<td>Gibco</td>
			<td>12100046</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-sheep conjugated with Alexa Fluor 568</td>
			<td>Abcam</td>
			<td>ab175712</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Cytiva</td>
			<td>SH3091003HI</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat antirabbit IgG labeled with Alexa Fluor 488&#160;</td>
			<td>Abcam</td>
			<td>ab150077</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution (HBSS)&#160;</td>
			<td>Gibco</td>
			<td>14175145</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Pancreatic Stellate Cells (HPaSteC)</td>
			<td>ScienCell</td>
			<td>3830</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Nikon</td>
			<td>Nikon</td>
			<td>Eclipse Ts 100</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc 96-Well Polystyrene Round Bottom Microwell Plates</td>
			<td>Thermo Scientific</td>
			<td>12-565-331</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Fluoview FV1200 confocal laser</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td>Discontinued product</td>
		</tr>
		<tr>
			<td>PANC-1</td>
			<td>ATCC</td>
			<td>CRL-1469</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-HEMA</td>
			<td>Sigma</td>
			<td>P3932</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti-laminin antibodies&#160;</td>
			<td>Abcam</td>
			<td>ab11575</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti-type I collagen antibodies&#160;</td>
			<td>Abcam</td>
			<td>ab34710</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheep polyclonal anti-hyaluronic acid antibodies</td>
			<td>Abcam</td>
			<td>ab53842</td>
			<td></td>
		</tr>
		<tr>
			<td>Stellate cell media complete kit&#160;</td>
			<td>ScienCell</td>
			<td>5301</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>MP Biomedicals, LLC</td>
			<td>153571</td>
			<td>Trypsin solution prepared according to manufacturers protocol and used at 0.25%w/v&#160;</td>
		</tr>
		<tr>
			<td>Trypsin Neutralization Solution (TNS)</td>
			<td>ScienCell</td>
			<td>103</td>
			<td></td>
		</tr>
	</tbody>
</table>

growing desmoplastic three-dimensional pancreatic cancer spheroids from co-culture

Pancreatic cancer&#39;s poor prognosis is associated with a variety of reasons, among which is its lack of easily detectable biomarkers leading to a late detection. Another major reason is the thick stroma surrounding the tissue, which leads to reduced blood supply. The deposition of large amounts of extracellular matrix (ECM), cell-cell interaction, endothelial cells, various immune cells, pericytes, proliferating myofibroblast, and fibroblast population, and the presence of non-neoplastic cells (together constituting the desmoplastic reaction)1, constitute the thick stroma that is responsible for PDAC&#39;s chemo and radiotherapeutic resistance2. Cancer and stromal cells have a complex, dynamic, and bidirectional interaction. Although some elements either attenuate or accelerate disease progression, most processes are adaptive during the tumor&#39;s development1. This provides an environment rich with growth factors, proangiogenic factors, proteases, and adhesion molecules. These factors promote angiogenesis, cell proliferation, metastasis, and invasion3,4. Together, they are immune and drug-privileged sanctuary for the tumor, resulting in drug resistance.
The desmoplasia is a complex mixture which consists of various ECM proteins, along with immune cells and pancreatic stellate cells (PSC). Together, these tend to form a scaffold for the cells to grow. PSCs are one of the largest components of the stromal compartment5. Their ability to produce enzymes like matrix metalloproteases (MMP), tissue inhibitors of matrix metalloproteases (TIMP) and cancer associated fibroblasts (CAF)6&#160;imply they are likely to play a critical role in development of the desmoplastic reaction. The ECM, cancer-associated fibroblasts (CAF), and vasculature are the cardinal aspects of PDAC. Among CAFs, myofibroblast and inflammatory CAFs are speculated to be involved in active crosstalk responsible for pro-tumor properties7. The more extensive the fibroblastic formations on the tumor, the poorer the prognosis8,9,10.
Monolayer cell culture through established cell lines continues to remain a useful tool for analyzing drug toxicity and is a good starting point for proof of concept and discovery studies. Established cell culture lines, however, lack germline DNA and clinical relatability11. Since they are grown on flat surfaces, they undergo different in vitro selection criteria compared to when they are a part of the tumor, divide abnormally and lose their differentiated phenotype12. Overall, single cell cultures limit tumor heterogeneity and therefore lose clinical relevance. They are unable to accurately represent the complexity of the tumor&#39;s microenvironment (for e.g., the ECM). 3D culture can more closely replicate the complex tumor microenvironment.
3D culture was introduced in the 1970&#39;s for healthy cells and their neoplastic counterparts13. Several techniques have been used to study the morphology and architecture of malignant tissues through spheroids14. Co-cultures with stromal cells can model TME signals. An upregulation of EMT markers was seen when cells were co-cultured with stellate cells15. PDAC spheroids and their interaction with the stroma can be modelled by co-culturing with ECM components. Co-culturing specifically with PSCs have been reported to produce clinically relevant drug cytotoxicity data16,17,18. PSCs also aid drug resistance by evading apoptosis and stimulating proliferation of cancer cells through various paracrine factors19&#160;and by inducing EMT transition. It, therefore, becomes critical to include the PSCs from an early stage in the criteria used to evaluate the success of a drug or drug delivery system. The PSC&#39;s ability to enhance proliferation and support faster growth in combination, compared to pancreatic cancer cells alone, has also been seen in vivo when subcutaneous flank injections of the two cell lines were evaluated in immunocompromised mice20.
The ability of a cell type to interact with ECM components is also critical to consider when growing co-cultured spheroids. BxPC-3 and PANC-1 have been reported to have equal affinities in binding to collagen. The two cell lines also bind equivalently to laminin, although there have been reports that BxPC-3 binds better21,22,23,24,25. In terms of migration, Stahle et al.26&#160;demonstrated a 5x faster motility for PANC-1 cells as compared to BxPC-3. PANC-1 cells were also reported to migrate primarily as single cells, whereas BxPC-3 cells migrate as a tightly packed sheet. The choice of the cells also affects the size of the tumor25. BxPC-3 tumors were shown to be larger27,28&#160;than those obtained from PANC-1, whereas one study demonstrated the opposite29&#160;case. Despite their differences in size and motility, both cells have been reported to need long periods of latency to form tumors in mice. This duration can be especially long for BxPC-3 ranging from 4 weeks to 4 months25. However, there is also literature where BxPC-328&#160;or BxPC-3 cancer stem cells30&#160;have formed visible tumors quicker, implying there could be variation seen in tumor growth durations. The durations stated here should, therefore, only serve as an initial guideline for tumor growth rates.
BxPC-3 cells form spheroids with loose cells on the surface and dense cores, whereas PANC-1 cells have been reported to form both porous but robust spheroids31 as well as compact spheroids. PANC-1 cells have also been reported to be less differentiated and more aggressive32. Keeping the aggressive nature32 at the forefront, combined with the PANC-1 cells&#39; higher motility, ability to form compact spheroids, and ability to interact with ECM components, PANC-1 cells were chosen for spheroid studies.
In the last few years, spheroid culture has seen a lot of success in demonstrating an advantage in its clinical relevancy compared to two-dimensional (2D) cultures. Its relevance has been leveraged in using this technique as a substitute to animal studies and to better understand the tumors&#39; biology. The clinical relevance of spheroids, especially when co-cultured with PSC&#39;s has enabled their use to study various functions of the spheroid such as stiffness33, expression of TGF-&#946;34,35,36,37,38, E-cadherin, F-actin18,34,36,37, &#945;-SMA34,35,37,38, lactate dehydrogenase (LDHA)32, HIF-1&#945;35,39, drug resistance16,37,40, cell migration41, cell invasion37, fibrosis35, radiation resistance42, phenotypical changes18, heterogeneity36, cellular levels of interactions39 and demonstrate ECM components37,38,39. Many of the protocols that were used to obtain the data described rely on Matrigel, the hanging drop method, printed molds, or other scaffolds to help support spheroid and ECM growth. The studies also usually involve the use of either non-human fibroblastic cells or freshly isolated stellate cells from patients. While using stellate cells is critical for the tumors to resemble in-vivo conditions, the inter-patient variability associated with fresh extractions makes these studies difficult to replicate.
This protocol aims to demonstrate a model that is easy to develop, reproducible, clinically relevant, and free of scaffolding, thereby relying exclusively on the co-cultures&#39; abilities to naturally generate the ECM. To do this, a simple co-culture method involving a mix of PANC-1 cells (due to their natural tendency to migrate as single cells) along with human pancreatic stellate cells (HPaSteC) was chosen, due to their ability to behave like stem cells and be highly drug-resistant. Using the studies by Durymanov et al.38as a baseline, the protocol detailed below was established after further optimizing parameters such as cell ratios and durations between media changes. The spheroids resulting from this protocol can be used as a model system for new drug candidate evaluation40.
Additionally, for users not familiar with the spheroid culture, the work Peirsman et al.43discussing the development of the MISpheroID knowledgebase may be helpful. It establishes some minimum information guidelines that could help cope with heterogeneity between lab protocols. Although with some limitations, the work demonstrated that the choice of culture media, cell lines, spheroid formation method, and the final spheroid size are critical in determining the phenotypic properties of spheroids.

<meta charset="utf8"></head><body>Auteur in de schijnwerpers: Validatie van reacties op kankertherapie met desmoplastische sferoïde modellen

Groeiende desmoplastische driedimensionale alvleesklierkankersferoïden uit co-cultuur

3D spheroid cultures are increasingly used to answer mechanistic questions about tumor growth and serve to screen new therapeutics. Therefore, it is critical that these models are clinically representative. We focus on replicating the desmoplasia seen in many cancers, including pancreatic cancer, using this technique.Spheroid cultures have evolved to incorporate patient-derived cells, complex co-cultures with immune cells, and physiological structures like vasculature. Additionally, microfluidic technology is employed for high-throughput screening and creating organ-on-a-chip systems. Multi-omic profiling in spheroids is now utilized to gain mechanistic insights.The described method can be used to create reproducible 3D co-cultured spheroids with a robust ECM free of synthetic matrix materials. The advantage is that the ECM closely replicates clinical tumors without their relevant synthetic matrix interfering with therapeutic screening applications. This model will enable the valuation of ECM response to therapeutics and answer important mechanism questions on the dynamic barrier that the ECM presents in numerous cancers.We plan to expand this model to include immune cell and macrophage components in addition to expanding the model used to understand ECM as a barrier to therapeutics and diseases outside of cancer such as tuberculosis granulomas.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of &#60;12%. The biggest barrier to therapy is the ...

growing-desmoplastic-three-dimensional-pancreatic-cancer-spheroids

Research

JoVE Journal

Cancer Research

Growing Desmoplastic Three-Dimensional Pancreatic Cancer Spheroids from Co-Culture

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of &#60;12%. The biggest barrier to therapy is the dense desmoplastic extracellular matrix (ECM) that surrounds the tumor and reduces vascularization, generally termed desmoplasia. A variety of drug combinations and formulations have been tested to treat the cancer, and although many of them show success pre-clinically, they fail clinically. It, therefore, becomes important to have a clinically relevant model available that can predict the response of the tumor to therapy. This model has been previously validated against resected clinical tumors. Here a simple protocol to grow desmoplastic three-dimensional (3D)-coculture spheroids is described that can naturally generating a robust ECM and do not require any external matrix sources or scaffold to support their growth.
Briefly human pancreatic stellate cells (HPaSteC) and PANC-1 cells are used to prepare a suspension containing the cells in a 1:2 ratio, respectively. The cells are plated in a poly-HEMA coated, 96-well low attachment U-well plate. The plate is centrifuged to allow the cells to form an initial pellet. The plate is stored in the incubator at 37 &#176;C with 5% CO2, and media is replaced every 3 days. Plates can be imaged at designated intervals to measure spheroid volume. Following 14 days of culture, mature desmoplastic spheroids are formed (i.e. average volume of 0.048 + 0.012 mm3&#160;(451 &#181;m x 462.84 &#181;m)) and can be utilized for experimental therapy assessment. Mature ECM components include collagen-I, hyaluronic acid, fibronectin, and laminin.

Pancreatic cancer remains one of the toughest cancers to treat. Therefore, it is critical that pre-clinical models evaluating treatment efficacy are reproducible and clinically relevant. This protocol describes a simple co-culture procedure to generate reproducible, clinically relevant desmoplastic spheroids.

Preparation of 3D Coculture Spheroids Mimicking Pancreatic Ductal Adenocarcinoma Desmoplasia for Therapy Testing

To begin, obtain PANC-1 cell culture and wash the cells with nine milliliters of Hank's balanced salt solution in three milliliter increments. Incubate the cells with two milliliters of trypsin for about 10 minutes. To neutralize the suspension, add three milliliters of DMEM supplemented with 10%FBS.After washing the cells seven times with one milliliter media increments, collect each neutralized aliquot in a 15 milliliter tube. Similarly, obtain trypsinized human pancreatic stellate cells, or HPaSteC, using trypsin neutralization solution and stellate cell media. Mix the required amount of both the cell suspensions in 11 milliliters of DMEM with 10%FBS avoiding froth formation.Gently dispense 100 microliters of cell suspension mix into the culture plate and incubated at 37 degrees Celsius with 5%carbon dioxide. On day three, add 50 microliters of DMEM with 10%FBS to each well along the side of the well. On day 10 or 11, aspirate the spent media without disturbing the solution near the halo and add 100 microliters of fresh media along the side of each well.On day 14 or 17, using a one milliliter pipette, remove media from each well until the halo is reached. Align the plate against the background of the hood to locate the spheroids at the bottom. Gently pipette around 50 microliters of media near the spheroids to disturb it for pooling.The spheroids obtained on day 14 were found to grow up to an average volume of 0.048 cubic millimeters.