Cancers

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18 pages, 5601 KiB

Open AccessArticle

Loss of Neuropilin-2 in Murine Mesenchymal-like Colon Cancer Organoids Causes Mesenchymal-to-Epithelial Transition and an Acquired Dependency on Insulin-Receptor Signaling and Autophagy

by Susanna Poghosyan, Nicola Frenkel, Aristeidis Lentzas, Jamila Laoukili, Inne Borel Rinkes, Onno Kranenburg and Jeroen Hagendoorn

Cancers 2022, 14(3), 671; https://doi.org/10.3390/cancers14030671 - 28 Jan 2022

Cited by 4 | Viewed by 2838

Abstract

Neuropilin-2 (Nrp2), an important regulator of lymphangiogenesis and lymphatic metastasis, has been associated with progression in colorectal cancer (CRC). However, the tumor cell-intrinsic role of Nrp2 in cancer progression is incompletely understood. To address this question, we employed CRISPR-Cas9 technology to generate Nrp2-knockout [...] Read more.

Neuropilin-2 (Nrp2), an important regulator of lymphangiogenesis and lymphatic metastasis, has been associated with progression in colorectal cancer (CRC). However, the tumor cell-intrinsic role of Nrp2 in cancer progression is incompletely understood. To address this question, we employed CRISPR-Cas9 technology to generate Nrp2-knockout organoids derived from murine CRC tumors with a mesenchymal phenotype. Transcriptome profiling and tumor tissue analysis showed that Nrp2 loss resulted in mesenchymal-to-epithelial transition (MET), which was accompanied with restored polarity and tight junction stabilization. Signaling pathway analysis revealed that Nrp2-knockout organoids acquire de novo dependency on insulin receptor (IR) signaling and autophagy as alternative survival mechanisms. Combined inhibition of IR signaling and autophagy prevented the stabilization of cell-cell junctions, reduced metabolic activity, and caused profound cell death in Nrp2-knockout organoids. Collectively, the data demonstrate a key role for Nrp2 in maintaining the aggressive phenotype and survival of tumor-derived CRC organoids. The identified connection between Nrp2, insulin receptor signaling and autophagy may guide the development of novel combination-treatment strategies for aggressive CRC. Full article

(This article belongs to the Special Issue Three-Dimensional Culture Systems in Cancer Research)

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27 pages, 3431 KiB

Open AccessEditor’s ChoiceArticle

The Anti-Proliferative Effect of PI3K/mTOR and ERK Inhibition in Monolayer and Three-Dimensional Ovarian Cancer Cell Models

by Elizabeth Dunn, Kenny Chitcholtan, Peter Sykes and Ashley Garrill

Cancers 2022, 14(2), 395; https://doi.org/10.3390/cancers14020395 - 13 Jan 2022

Cited by 9 | Viewed by 2718

Abstract

Most ovarian cancer patients are diagnosed with advanced stage disease, which becomes unresponsive to chemotherapeutic treatments. The PI3K/AKT/mTOR and the RAS/RAF/MEK/ERK kinase signaling pathways are attractive targets for potential therapeutic inhibitors, due to the high frequency of mutations to PTEN, PIK3CA, KRAS and [...] Read more.

Most ovarian cancer patients are diagnosed with advanced stage disease, which becomes unresponsive to chemotherapeutic treatments. The PI3K/AKT/mTOR and the RAS/RAF/MEK/ERK kinase signaling pathways are attractive targets for potential therapeutic inhibitors, due to the high frequency of mutations to PTEN, PIK3CA, KRAS and BRAF in several ovarian cancer subtypes. However, monotherapies targeting one of these pathways have shown modest effects in clinical trials. This limited efficacy of the agents could be due to upregulation and increased signaling via the adjacent alternative pathway. In this study, the efficacy of combined PI3K/mTOR (BEZ235) and ERK inhibition (SCH772984) was investigated in four human ovarian cancer cell lines, grown as monolayer and three-dimensional cell aggregates. The inhibitor combination reduced cellular proliferation in a synergistic manner in OV-90 and OVCAR8 monolayers and in OV-90, OVCAR5 and SKOV3 aggregates. Sensitivity to the inhibitors was reduced in three-dimensional cell aggregates in comparison to monolayers. OV-90 cells cultured in large spheroids were sensitive to the inhibitors and displayed a robust synergistic antiproliferative response to the inhibitor combination. In contrast, OVCAR8 spheroids were resistant to the inhibitors. These findings suggest that combined PI3K/mTOR and ERK inhibition could be a useful strategy for overcoming treatment resistance in ovarian cancer and warrants further preclinical investigation. Additionally, in some cell lines the use of different three-dimensional models can influence cell line sensitivity to PI3K/mTOR and RAS/RAF/MEK/ERK pathway inhibitors. Full article

(This article belongs to the Special Issue Three-Dimensional Culture Systems in Cancer Research)

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12 pages, 2993 KiB

Open AccessArticle

Mismatch Repair Status in Patient-Derived Colorectal Cancer Organoids Does Not Affect Intrinsic Tumor Cell Sensitivity to Systemic Therapy

by Emre Küçükköse, G. Emerens Wensink, Celine M. Roelse, Susanne J. van Schelven, Daniëlle A. E. Raats, Sylvia F. Boj, Miriam Koopman, Jamila Laoukili, Jeanine M. L. Roodhart and Onno Kranenburg

Cancers 2021, 13(21), 5434; https://doi.org/10.3390/cancers13215434 - 29 Oct 2021

Cited by 4 | Viewed by 2533

Abstract

DNA mismatch repair deficiency (dMMR) in metastatic colorectal cancer (mCRC) is associated with poor survival and a poor response to systemic treatment. However, it is unclear whether dMMR results in a tumor cell-intrinsic state of treatment resistance, or whether alternative mechanisms play a [...] Read more.

DNA mismatch repair deficiency (dMMR) in metastatic colorectal cancer (mCRC) is associated with poor survival and a poor response to systemic treatment. However, it is unclear whether dMMR results in a tumor cell-intrinsic state of treatment resistance, or whether alternative mechanisms play a role. To address this, we generated a cohort of MMR-proficient and -deficient Patient-Derived Organoids (PDOs) and tested their response to commonly used drugs in the treatment of mCRC, including 5-fluorouracil (5-FU), oxaliplatin, SN-38, binimetinib, encorafenib, and cetuximab. MMR status did not correlate with the response of PDOs to any of the drugs tested. In contrast, the presence of activating mutations in the KRAS and BRAF oncogenes was significantly associated with resistance to chemotherapy and sensitivity to drugs targeting oncogene-activated pathways. We conclude that mutant KRAS and BRAF impact the intrinsic sensitivity of tumor cells to chemotherapy and targeted therapy. By contrast, tumor cell-extrinsic mechanisms—for instance signals derived from the microenvironment—must underlie the association of MMR status with therapy response. Future drug screens on rationally chosen cohorts of PDOs have great potential in developing tailored therapies for specific CRC subtypes including, but not restricted to, those defined by BRAF/KRAS and MMR status. Full article

(This article belongs to the Special Issue Three-Dimensional Culture Systems in Cancer Research)

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29 pages, 22585 KiB

Open AccessArticle

Polycaprolactone Electrospun Scaffolds Produce an Enrichment of Lung Cancer Stem Cells in Sensitive and Resistant EGFRm Lung Adenocarcinoma

by Emma Polonio-Alcalá, Marc Rabionet, Santiago Ruiz-Martínez, Sònia Palomeras, Rut Porta, Carmen Vásquez-Dongo, Joaquim Bosch-Barrera, Teresa Puig and Joaquim Ciurana

Cancers 2021, 13(21), 5320; https://doi.org/10.3390/cancers13215320 - 22 Oct 2021

Cited by 4 | Viewed by 2685

Abstract

The establishment of a three-dimensional (3D) cell culture model for lung cancer stem cells (LCSCs) is needed because the study of these stem cells is unable to be done using flat surfaces. The study of LCSCs is fundamental due to their key role [...] Read more.

The establishment of a three-dimensional (3D) cell culture model for lung cancer stem cells (LCSCs) is needed because the study of these stem cells is unable to be done using flat surfaces. The study of LCSCs is fundamental due to their key role in drug resistance, tumor recurrence, and metastasis. Hence, the purpose of this work is the evaluation of polycaprolactone electrospun (PCL-ES) scaffolds for culturing LCSCs in sensitive and resistant EGFR-mutated (EGFRm) lung adenocarcinoma cell models. We performed a thermal, physical, and biological characterization of 10% and 15%-PCL-ES structures. Several genes and proteins associated with LCSC features were analyzed by RT-qPCR and Western blot. Vimentin and CD133 tumor expression were evaluated in samples from 36 patients with EGFRm non-small cell lung cancer through immunohistochemistry. Our findings revealed that PC9 and PC9-GR3 models cultured on PCL-ES scaffolds showed higher resistance to osimertinib, upregulation of ABCB1, Vimentin, Snail, Twist, Sox2, Oct-4, and CD166, downregulation of E-cadherin and CD133, and the activation of Hedgehog pathway. Additionally, we determined that the non-expression of CD133 was significantly associated with a low degree of histological differentiation, disease progression, and distant metastasis. To sum up, we confirmed PCL-ES scaffolds as a suitable 3D cell culture model for the study of the LCSC niche. Full article

(This article belongs to the Special Issue Three-Dimensional Culture Systems in Cancer Research)

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23 pages, 3383 KiB

Open AccessArticle

Hyaluronic Acid-Functionalized Nanomicelles Enhance SAHA Efficacy in 3D Endometrial Cancer Models

by Kadie Edwards, Seydou Yao, Simone Pisano, Veronica Feltracco, Katja Brusehafer, Sumanta Samanta, Oommen P. Oommen, S. Andrea Gazze, Roberta Paravati, Holly Maddison, Chao Li, Deyarina Gonzalez, R. Steven Conlan and Lewis Francis

Cancers 2021, 13(16), 4032; https://doi.org/10.3390/cancers13164032 - 10 Aug 2021

Cited by 9 | Viewed by 4742

Abstract

Histone Deacetylase (HDAC) enzymes are upregulated in cancer leading to the development of HDAC inhibiting compounds, several of which are currently in clinical trials. Side effects associated with toxicity and non-specific targeting indicate the need for efficient drug delivery approaches and tumor specific [...] Read more.

Histone Deacetylase (HDAC) enzymes are upregulated in cancer leading to the development of HDAC inhibiting compounds, several of which are currently in clinical trials. Side effects associated with toxicity and non-specific targeting indicate the need for efficient drug delivery approaches and tumor specific targeting to enhance HDAC efficacy in solid tumor cancers. SAHA encapsulation within F127 micelles functionalized with a surface hyaluronic acid moiety, was developed to target endometrial cancer cells expressing elevated levels of CD44. In vitro viability and morphology analyses was conducted in both 2D and 3D models to assess the translational potential of this approach. Encapsulation enhanced SAHA delivery and activity, demonstrating increased cytotoxic efficacy in 2D and 3D endometrial cancer models. High-content imaging showed improved nanoparticle internalization in 2D and CD44 enhanced penetration in 3D models. In addition, the nano-delivery system enhanced spheroid penetration resulting in cell growth suppression, p21 associated cell cycle arrest, as well as overcoming the formation of an EMT associated phenotype observed in free drug treated type II endometrial cancer cells. This study demonstrates that targeted nanoparticle delivery of SAHA could provide the basis for improving its efficacy in endometrial cancer. Using 3D models for endometrial cancer allows the elucidation of nanoparticle performance and CD44 targeting, likely through penetration and retention within the tumor model. Full article

(This article belongs to the Special Issue Three-Dimensional Culture Systems in Cancer Research)

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19 pages, 7099 KiB

Open AccessArticle

Loss of Nitric Oxide Induces Fibrogenic Response in Organotypic 3D Co-Culture of Mammary Epithelia and Fibroblasts—An Indicator for Breast Carcinogenesis

by Gang Ren, Xunzhen Zheng, Vandana Sharma, Joshua Letson, Andrea L. Nestor-Kalinoski and Saori Furuta

Cancers 2021, 13(11), 2815; https://doi.org/10.3390/cancers13112815 - 5 Jun 2021

Cited by 2 | Viewed by 3047

Abstract

Excessive myofibroblast activation, which leads to dysregulated collagen deposition and the stiffening of the extracellular matrix (ECM), plays pivotal roles in cancer initiation and progression. Cumulative evidence attests to the cancer-causing effects of a number of fibrogenic factors found in the environment, diseases [...] Read more.

Excessive myofibroblast activation, which leads to dysregulated collagen deposition and the stiffening of the extracellular matrix (ECM), plays pivotal roles in cancer initiation and progression. Cumulative evidence attests to the cancer-causing effects of a number of fibrogenic factors found in the environment, diseases and drugs. While identifying such factors largely depends on epidemiological studies, it would be of great importance to develop a robust in vitro method to demonstrate the causal relationship between fibrosis and cancer. Here, we tested whether our recently developed organotypic three-dimensional (3D) co-culture would be suitable for that purpose. This co-culture system utilizes the discontinuous ECM to separately culture mammary epithelia and fibroblasts in the discrete matrices to model the complexity of the mammary gland. We observed that pharmaceutical deprivation of nitric oxide (NO) in 3D co-cultures induced myofibroblast differentiation of the stroma as well as the occurrence of epithelial–mesenchymal transition (EMT) of the parenchyma. Such in vitro response to NO deprivation was unique to co-cultures and closely mimicked the phenotype of NO-depleted mammary glands exhibiting stromal desmoplasia and precancerous lesions undergoing EMT. These results suggest that this novel 3D co-culture system could be utilized in the deep mechanistic studies of the linkage between fibrosis and cancer. Full article

(This article belongs to the Special Issue Three-Dimensional Culture Systems in Cancer Research)

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20 pages, 6189 KiB

Open AccessArticle

The New Serum-Free OptiPASS^® Medium in Cold and Oxygen-Free Conditions: An Innovative Conservation Method for the Preservation of MDA-MB-231 Triple Negative Breast Cancer Spheroids

by Antoine Goisnard, Clémence Dubois, Pierre Daumar, Corinne Aubel, Marie Depresle, Jean Gauthier, Bernard Vidalinc, Frédérique Penault-Llorca, Emmanuelle Mounetou and Mahchid Bamdad

Cancers 2021, 13(8), 1945; https://doi.org/10.3390/cancers13081945 - 18 Apr 2021

Cited by 2 | Viewed by 2470

Abstract

Cancer spheroids are very effective preclinical models to improve anticancer drug screening. In order to optimize and extend the use of spheroid models, these works were focused on the development of a new storage concept to maintain these models in the longer term [...] Read more.

Cancer spheroids are very effective preclinical models to improve anticancer drug screening. In order to optimize and extend the use of spheroid models, these works were focused on the development of a new storage concept to maintain these models in the longer term using the Triple-Negative Breast Cancer MDA-MB-231 spheroid models. The results highlight that the combination of a temperature of 4 °C and oxygen-free conditions allowed the spheroid characteristics of OptiPASS^® serum-free culture medium to preserve the spheroid characteristics during 3-, 5- or 7-day-long storage. Indeed, after storage they were returned to normal culture conditions, with recovered spheroids presenting similar growth rates (recovery = 96.2%), viability (Live/Dead^® profiles) and metabolic activities (recovery = 90.4%) compared to nonstored control spheroids. Likewise, both recovered spheroids (after storage) and nonstored controls presented the same response profiles as two conventional drugs, i.e., epirubicin and cisplatin, and two anti-PARP1 targeted drugs—i.e., olaparib and veliparib. This new original storage concept seems to induce a temporary stop in spheroid growth while maintaining their principal characteristics for further use. In this way, this innovative and simple storage concept may instigate future biological sample preservation strategies. Full article

(This article belongs to the Special Issue Three-Dimensional Culture Systems in Cancer Research)

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18 pages, 2831 KiB

Open AccessArticle

PDX-Derived Ewing’s Sarcoma Cells Retain High Viability and Disease Phenotype in Alginate Encapsulated Spheroid Cultures

by Giacomo Domenici, Rodrigo Eduardo, Helena Castillo-Ecija, Gorka Orive, Ángel Montero Carcaboso and Catarina Brito

Cancers 2021, 13(4), 879; https://doi.org/10.3390/cancers13040879 - 19 Feb 2021

Cited by 5 | Viewed by 4044

Abstract

Ewing’s Sarcoma (ES) is the second most frequent malignant bone tumour in children and young adults and currently only untargeted chemotherapeutic approaches and surgery are available as treatment, although clinical trials are on-going for recently developed ES-targeted therapies. To study ES pathobiology and [...] Read more.

Ewing’s Sarcoma (ES) is the second most frequent malignant bone tumour in children and young adults and currently only untargeted chemotherapeutic approaches and surgery are available as treatment, although clinical trials are on-going for recently developed ES-targeted therapies. To study ES pathobiology and develop novel drugs, established cell lines and patient-derived xenografts (PDX) are the most employed experimental models. Nevertheless, the establishment of ES cell lines is difficult and the extensive use of PDX raises economic/ethical concerns. There is a growing consensus regarding the use of 3D cell culture to recapitulate physiological and pathophysiological features of human tissues, including drug sensitivity. Herein, we implemented a 3D cell culture methodology based on encapsulation of PDX-derived ES cell spheroids in alginate and maintenance in agitation-based culture systems. Under these conditions, ES cells displayed high proliferative and metabolic activity, while retaining the typical EWSR1-FLI1 chromosomal translocation. Importantly, 3D cultures presented reduced mouse PDX cell contamination compared to 2D cultures. Finally, we show that these 3D cultures can be employed in drug sensitivity assays, with results similar to those reported for the PDX of origin. In conclusion, this novel 3D cell culture method involving ES-PDX-derived cells is a suitable model to study ES pathobiology and can assist in the development of novel drugs against this disease, complementing PDX studies. Full article

(This article belongs to the Special Issue Three-Dimensional Culture Systems in Cancer Research)

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Review

Jump to: Research, Other

16 pages, 1060 KiB

Open AccessReview

In Vitro 3D Cultures to Model the Tumor Microenvironment

by Fabrizio Fontana, Monica Marzagalli, Michele Sommariva, Nicoletta Gagliano and Patrizia Limonta

Cancers 2021, 13(12), 2970; https://doi.org/10.3390/cancers13122970 - 13 Jun 2021

Cited by 43 | Viewed by 5728

Abstract

It is now well established that the tumor microenvironment plays a key role in determining cancer growth, metastasis and drug resistance. Thus, it is fundamental to understand how cancer cells interact and communicate with their stroma and how this crosstalk regulates disease initiation [...] Read more.

It is now well established that the tumor microenvironment plays a key role in determining cancer growth, metastasis and drug resistance. Thus, it is fundamental to understand how cancer cells interact and communicate with their stroma and how this crosstalk regulates disease initiation and progression. In this setting, 3D cell cultures have gained a lot of interest in the last two decades, due to their ability to better recapitulate the complexity of tumor microenvironment and therefore to bridge the gap between 2D monolayers and animal models. Herein, we present an overview of the 3D systems commonly used for studying tumor–stroma interactions, with a focus on recent advances in cancer modeling and drug discovery and testing. Full article

(This article belongs to the Special Issue Three-Dimensional Culture Systems in Cancer Research)

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24 pages, 1632 KiB

Open AccessReview

Organoids and Colorectal Cancer

by Antonio Barbáchano, Asunción Fernández-Barral, Pilar Bustamante-Madrid, Isabel Prieto, Nuria Rodríguez-Salas, María Jesús Larriba and Alberto Muñoz

Cancers 2021, 13(11), 2657; https://doi.org/10.3390/cancers13112657 - 28 May 2021

Cited by 27 | Viewed by 8251

Abstract

Organoids were first established as a three-dimensional cell culture system from mouse small intestine. Subsequent development has made organoids a key system to study many human physiological and pathological processes that affect a variety of tissues and organs. In particular, organoids are becoming [...] Read more.

Organoids were first established as a three-dimensional cell culture system from mouse small intestine. Subsequent development has made organoids a key system to study many human physiological and pathological processes that affect a variety of tissues and organs. In particular, organoids are becoming very useful tools to dissect colorectal cancer (CRC) by allowing the circumvention of classical problems and limitations, such as the impossibility of long-term culture of normal intestinal epithelial cells and the lack of good animal models for CRC. In this review, we describe the features and current knowledge of intestinal organoids and how they are largely contributing to our better understanding of intestinal cell biology and CRC genetics. Moreover, recent data show that organoids are appropriate systems for antitumoral drug testing and for the personalized treatment of CRC patients. Full article

(This article belongs to the Special Issue Three-Dimensional Culture Systems in Cancer Research)

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25 pages, 9997 KiB

Open AccessReview

In Vitro Glioblastoma Models: A Journey into the Third Dimension

by Mayra Paolillo, Sergio Comincini and Sergio Schinelli

Cancers 2021, 13(10), 2449; https://doi.org/10.3390/cancers13102449 - 18 May 2021

Cited by 32 | Viewed by 5940

Abstract

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor in adults, with an average survival time of about one year from initial diagnosis. In the attempt to overcome the complexity and drawbacks associated with in vivo GBM models, together with the need [...] Read more.

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor in adults, with an average survival time of about one year from initial diagnosis. In the attempt to overcome the complexity and drawbacks associated with in vivo GBM models, together with the need of developing systems dedicated to screen new potential drugs, considerable efforts have been devoted to the implementation of reliable and affordable in vitro GBM models. Recent findings on GBM molecular features, revealing a high heterogeneity between GBM cells and also between other non-tumor cells belonging to the tumoral niche, have stressed the limitations of the classical 2D cell culture systems. Recently, several novel and innovative 3D cell cultures models for GBM have been proposed and implemented. In this review, we first describe the different populations and their functional role of GBM and niche non-tumor cells that could be used in 3D models. An overview of the current available 3D in vitro systems for modeling GBM, together with their major weaknesses and strengths, is presented. Lastly, we discuss the impact of groundbreaking technologies, such as bioprinting and multi-omics single cell analysis, on the future implementation of 3D in vitro GBM models. Full article

(This article belongs to the Special Issue Three-Dimensional Culture Systems in Cancer Research)

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Other

Jump to: Research, Review

18 pages, 1631 KiB

Open AccessSystematic Review

Dynamic Physiological Culture of Ex Vivo Human Tissue: A Systematic Review

by Daniel Ll Hughes, Aron Hughes, Zahir Soonawalla, Somnath Mukherjee and Eric O’Neill

Cancers 2021, 13(12), 2870; https://doi.org/10.3390/cancers13122870 - 8 Jun 2021

Cited by 11 | Viewed by 2979

Abstract

Conventional static culture fails to replicate the physiological conditions that exist in vivo. Recent advances in biomedical engineering have resulted in the creation of novel dynamic culturing systems that permit the recapitulation of normal physiological processes ex vivo. Whilst the physiological benefit for [...] Read more.

Conventional static culture fails to replicate the physiological conditions that exist in vivo. Recent advances in biomedical engineering have resulted in the creation of novel dynamic culturing systems that permit the recapitulation of normal physiological processes ex vivo. Whilst the physiological benefit for its use in the culture of two-dimensional cellular monolayer has been validated, its role in the context of primary human tissue culture has yet to be determined. This systematic review identified 22 articles that combined dynamic physiological culture techniques with primary human tissue culture. The most frequent method described (55%) utilised dynamic perfusion culture. A diverse range of primary human tissue was successfully cultured. The median duration of successful ex vivo culture of primary human tissue for all articles was eight days; however, a wide range was noted (5 h–60 days). Six articles (27%) reported successful culture of primary human tissue for greater than 20 days. This review illustrates the physiological benefit of combining dynamic culture with primary human tissue culture in both long-term culture success rates and preservation of native functionality of the tissue ex vivo. Further research efforts should focus on developing precise biochemical sensors that would allow for real-time monitoring and automated self-regulation of the culture system in order to maintain homeostasis. Combining these techniques allows the creation of an accurate system that can be used to gain a greater understanding of human physiology. Full article

(This article belongs to the Special Issue Three-Dimensional Culture Systems in Cancer Research)

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