Experimental models of colorectal cancer

November 03, 2016 By: Rui Castro

© Can Stock Photo/focalpoint 

Dr Rui Castro is currently a Principal Investigator at the Research Institute for Medicines (iMed.ULisboa), Portugal. He completed his PhD at the University of Lisbon and the Department of Medicine (GI Division), University of Minnesota Medical School, USA, in 2006. Since then, Dr Castro has been combining his background on the modulation of liver cell function with his most recent discoveries in the miRNA field, to answer key questions on liver physiology and pathophysiology, while supervising both undergraduate and postgraduate students under the GI umbrella. In 2015, he was selected as a UEG Rising Star. Follow Rui on Twitter @RuiCastroHD.

Experimental models of colorectal cancer

Can you pinpoint hot trends in colorectal cancer research? 

Worldwide, colorectal cancer (CRC) is the third most frequent cancer in men and the second most frequent in women. In Europe, CRC has the highest cancer mortality rates for both sexes and, by 2020, it is estimated that there will be almost 50,000 new cases of CRC.Novel therapeutic approaches to CRC are urgently needed; surgical resection of tumours remains the best strategy to improve survival of patients, but more than half of them will go on to develop metastasis. In addition, currently available adjuvant chemotherapy for more advanced stages of CRC only benefit a small percentage of patients, mostly due to drug resistance and/or poor efficacy, and severe side effects.

 

The high incidence and mortality of CRC, together with the need for improved or new therapeutic options, have fuelled an increase in translational research worldwide. Indeed, significant advances have been made in the development of appropriate experimental models for CRC research, from in vitro to in vivo transplant models, as well as carcinogen-induced and genetically engineered animal models. Of note, the subject of the UEG Basic Science Course 2016 was “Hot topics in experimental GI cancer,” highlighting the current relevance of GI cancer research—with a heavy focus on hands-on training, there is no better way to learn about ground-breaking methodological approaches and techniques!

 

Of course, it is envisaged that no single model should be employed when studying the pathogenesis and potential chemotherapeutic and biological treatments of CRC (or any disease for that matter). Having said that, one particular model is currently seen as a hot topic, owing to its ability to model many of the genetic alterations associated with CRC carcinogenesis (ease of genetic manipulation) and its capacity to act as a model system for both basic and translational research, including high-throughput testing of individual responses to both existing and potential new drugs—so-called personalized medicine.

CAN YOU PINPOINT WHICH MODEL THIS IS?

a)         ApcMin/+ mouse model

b)         Organoid culture system model

c)         Patient derived xenograft (PDX) model 

d)         Syngraft/isograft model

Answer

Correct answer: b.

Discussion

Most CRCs develop from a series of genetic and/or epigenetic alterations in the epithelium, leading to the formation of adenomas. Inactivation of the adenomatosis polyposis coli (Apc) gene is present in more than 80% of CRCs.2,3 The multiple intestinal neoplasia (ApcMin/+) mouse, which carries a truncation mutation at codon 850 of the Apc gene, was the first key model of CRC. Like most genetically engineered mouse models, the ApcMin/+ mouse model has been largely used in studies of CRC development and chemoprevention, as part of a translational approach. However, its use in preclinical drug discovery is very limited. In addition, the short lifespan of the ApcMin/+ mouse and the low metastatic rate make this model unsuitable for mimicking advanced CRC.4 Still, in recent years, many different Apc mutant mice with germline or inducible conditional alleles of Apc have been developed, such as the ApcCKO/CKO-LSL-Kras mice (G12D; Krastm4tyj/+ allele), which exhibit simultaneous inactivation of Apc and activation of Kras in the adult colon, leading to the formation of distant metastases.3,5 These genetically modified Apc mouse models allow for more accurate preclinical investigations, including screening for early disease biomarkers.

The establishment of 3D culture systems from intestinal stem cells for the growth and maintenance of tissues explanted from intestinal and colonic mouse and human samples is thought of by many as the next big thing in CRC research.6 The method for creating and maintaining self-renewing intestinal organoids was pioneered by Sato and co-workers in 20097 and has since been optimized.8,9 There are several useful applications of organoid systems in CRC research:6

  • Performing stem cell assays—functional evaluation of the stem cell capacity of distinct cell populations.


  • Easy genetic manipulation—sequential deletion of a series of genes, as a way to model the multistep CRC development process, is possible by using the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated 9 (Cas9) system. For instance, isogenic human organoids that model the adenoma–carcinoma sequence in human CRC can be generated by editing of Apc by CRISPR–Cas9, followed by deletion of SMAD4 and p53 and, further, by introduction of activating mutations in KRAS and PIK3CA.10,11,12 Furthermore, modelling the genetic alterations associated with CRC carcinogenesis also allows genetic events associated with CRC stem cell generation to be studied.


  • Studying poorly explored interactions between different cell types—it is possible to co-culture organoids from intestinal or colorectal adenomas with different cell types, such as lymphocytes, nerves and fibroblasts.


  • Comparing drug or chemotherapeutic responses between tumour spheroids and normal intestinal epithelial organoids, as a readout of drug efficacy. Furthermore, such testing can easily be scaled up to high-throughput screening (HTS) of drugs, while patient-derived organoids allow for personalized therapy assays, like testing for individual responses to both existing and potential new drugs. This type of testing is of particular value for patients carrying rare mutations. Last but not least, organoids can theoretically be cultured from different regions of the CRC tumour, to model tumour heterogeneity, and from primary and metastatic locations to study mechanisms of cancer metastasis.10

 

Patient derived xenografts (PDXs), in which portions of a patient’s tumour tissue are implanted into an immunodeficient mouse, preserve, at least temporarily, the patient’s specific tumour–stromal-cell interactions. As such, PDXs were seen as a promising way to test therapies ahead of patient treatment, to screen for efficacy and resistance. However, although PDXs may predict clinical response to therapy better than traditional xenografts, they also have several disadvantages. For a start, host cells appear to replace the human stroma and vasculature much more quickly than initially estimated. In addition, the model requires subculture or serial transplants, which is expensive, labour intensive and time consuming (requiring ≥6 months).3 Furthermore, similarly to traditional xenografts, PDXs still need to be implanted into immunodeficient host animals, while its tumour initiation rate has been suggested to be ~70%, further limiting clinical utility.10,13


A syngraft or isograft model involves the implantation of cell lines or tumour fragments derived from a particular species into an immunocompetent animal of the same strain. The biggest advantage of such a model is its ability to test immunotherapies. In fact, the role of the immune system in CRC development appears to be particularly important.14 As an example, Bindea and co-workers recently demonstrated that for CRC the CXCL13 chemokine positively correlates with disease-free survival; they then placed MC38 cells (mouse colon adenocarcinoma-derived cells) in mice lacking the CXCL13 receptor CXCR5. Strikingly, MC38 isografts were more proliferative and displayed higher growth rates in Cxcr5–/– mice than in wild-type animals, underscoring the prognostic value of CXCL13 in assessing CRC tumour burden.15 Furthermore, the rodents used in isograft models are cheaper and more robust compared with their immunodeficient counterparts. Still, it should be noted that tumours in this model will express the rodent homologues of human tumour genes, which may limit the testing of targeted therapies.4

References

  1. International Agency for Research on Cancer. GLOBOCAN 2012: Cancer incidence, mortality and prevalence worldwide. Downloaded on October 03 2016, from [http://globocan.iarc.fr/old/burden.asp?selection_pop=62968&Text-p=Europe&selection_cancer=5060&Text-c=Colorectal+cancer&pYear=8&type=0&window=1&submit=%C2%A0Execute%C2%A0]
  2. Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature 2012; 487: 330–337.
  3. McIntyre RE, Buczacki SJ, Arends MJ, et al. Mouse models of colorectal cancer as preclinical models. Bioessays 2015; 37: 909–920.
  4. Evans JP, Sutton PA, Winiarski BK, et al. From mice to men: Murine models of colorectal cancer for use in translational research. Crit Rev Oncol Hematol 2016; 98: 94–105.
  5. Hung KE, Maricevich MA, Richard LG, et al. Development of a mouse model for sporadic and metastatic colon tumors and its use in assessing drug treatment. Proc Natl Acad Sci USA 2010; 107: 1565–1570. 
  6. Young M and Reed KR. Organoids as a model for colorectal cancer. Curr Colorectal Cancer Rep 2016; 12: 281–287.
  7. Sato T, Vries RG, Snippert HJ, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 2009; 459: 262–265.
  8. Sato T, Stange DE, Ferrante M, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 2011; 141: 1762–1772.
  9. Jung P, Sato T, Merlos-Suárez A, et al. Isolation and in vitro expansion of human colonic stem cells. Nat Med 2011; 17: 1225–1227.
  10. Golovko D, Kedrin D, Yilmaz ÖH, et al. Colorectal cancer models for novel drug discovery. Expert Opin Drug Discov 2015; 10: 1217–1229.
  11. Matano M, Date S, Shimokawa M, et al. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat Med 2015; 21: 256–262.
  12. Drost J, van Jaarsveld RH, Ponsioen B, et al. Sequential cancer mutations in cultured human intestinal stem cells. Nature 2015; 521: 43–47.
  13. Aparicio S, Hidalgo M and Kung AL. Examining the utility of patient-derived xenograft mouse models. Nat Rev Cancer 2015; 15: 311–316.
  14. Galon J, Costes A, Sanchez-Cabo F, et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 2006; 313: 1960–1964.
  15. Bindea G, Mlecnik B, Tosolini M, et al. Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer. Immunity 2013; 39: 782–795.

 

Further UEG Resources

About the author

Dr Rui Castro is currently a Principal Investigator at the Research Institute for Medicines (iMed.ULisboa), Portugal. He completed his PhD at the University of Lisbon and the Department of Medicine (GI Division), University of Minnesota Medical School, USA, in 2006. Since then, Dr Castro has been combining his background on the modulation of liver cell function with his most recent discoveries in the miRNA field, to answer key questions on liver physiology and pathophysiology, while supervising both undergraduate and postgraduate students under the GI umbrella. In 2015, he was selected as a UEG Rising Star. Follow Rui on Twitter @RuiCastroHD.

 

Comments

Irina Korytko, November 14, 2016 11:59
A
Jarek Kobiela, November 03, 2016 23:56
Let it B!
Jarek Kobiela, November 03, 2016 23:49
I vote B!
Diane Pereira, November 03, 2016 16:16
B!!!

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