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Materials and methods

Slice cultures are thin cuts of the original tumor tissue, preserving the original morphology and cellular composition of tumors for several days up to weeks in culture, depending on the tumor entity Figure 1a-d. While spheroid models are widely described and recently reviewed for intestinal cancers [1] and others [2], we focus here on a brief overview of the slice culture model and describe future possibilities in this field. Figure 1: Human tumor slice cultures. Only the lower side of the tissue is in contact with the culture media, the upper side stays exposed to air.

Adjacent tissue was cultured for 24h with Isolectin B4 labelled and was then transferred to the tumor slice culture without macrophage labelling. Tissue was live imaged by a confocal microscope with a stable incubation chamber and a 20x objective. Tumor slice cultures can be useful for studying complex cellular processes and tumor-stroma interaction ex-vivo. In it was already shown that antibodies against a cell-surface receptor altered the growth pattern of cancerous cells kept in 3D cultures but not in monolayer cultures [3].

Since then, numerous changes were discovered and novel systems became established reviewed in [4,5].

Spheroids in cancer research. Methods and perspectives.

It became clear that tumor tissue consists of various cancer cell clones, influencing each other. These compilation needs to be maintained similar to the physiological condition, in order to compare tissue response in culture with the tumor response of the organism [6,7]. Intriguing papers were published that report about investigation and refinement of various technological issues. Vaira et al. Colon cancer tissue of the same patients was used to compare slice cultures ex vivo with primary monolayer cultures by microarray analysis. Colon tumor slice cultures showed little alterations whereas primary cultures were not comparable to the uncultured tumor tissue.

However, an important point to consider in this comparison is the different culture media used in the compared methods [9]. Moreover, promising novel therapeutics and mechanisms of resistance can be tested on human tumor slice cultures, guiding and potentially even preventing animal experiments. Animal research in cancer has profound impact on the quality of life of the animals and they may lead to severe suffering and death of the animal.


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Besides mutational mouse models, xenograft models are scientifically questionable in the context of tumor-stroma interaction. These mouse models are the golden standard in oncology research, yet they should be used with care as not only a missing immune-system but also species differences need to be considered. Fatal cytokine storms were developed by all human volunteers taking part in a clinical phase I trial.

The investigational compound, a monoclonal antibody directed against Cluster-of-differentiation CD 28 did not indicate any of these effects in non-human chimpanzees or mouse models. CD28 is one of the proteins expressed on human T cells that provide co-stimulatory signals required for T cell activation and survival. In contrast to the animal models, the reason for the observed severe clinical effect was reproducible in human lymphatic tissue slice cultures from the palentine tonsil.

The obtained slices were treated with the monoclonal antibody against CD28 and the culture media was monitored for cytokine release for 72h unpublished. Later, the antibody was identified to be a super-agonist in humans and a clinical trial using very small doses indicates now first signs of clinical efficacy, when applied in patients with rheumatoid arthritis [10]. In summary, the human tumor slice culture model is an enhanced model to study tumor heterogeneity ex-vivo and has the potential to reduce the translational step towards clinical studies.

Until today, slice cultures have mainly been used and are mainly published to test susceptibility to anti-cancer drugs []. In Glioblastoma slice cultures, the clinical O 6 -methylguanine-DNA methyltransferase MGMT promoter methylation did not reflect tumor slice culture response to clinical standard treatment.

Human tumor slice cultures for cancer research and drug testing

This, however, is also seen in clinical observations [11]. Further studies investigated tumor susceptibility in tumor slice cultures, however, the promising results are not yet clinically approved [8,12,13,15]. Thus, conserving tissue architecture and cellular diversity ex-vivo tumor slice culture promises to be a representative tumor model.

The different aspects of these approaches should be carefully considered and future research will demonstrate advantages and disadvantages of each set-up. One major advantage of tumor slice cultures on membranes is the possibility to image cellular activities live with a confocal microscope Figure 1e-g.

Studies using membranes to cultivate tumor slice cultures on a liquid-air interface could keep glioblastoma tissue slice cultures 16 days in culture [11]. Gastric cancer, colon cancer, head and neck tumor and non-small lung cancer slice cultures are well maintained up to six or seven days ex vivo [12,13].

Introduction

Even if the tissue is damaged and inflammatory processes are provoked at the surface of the tissue slice, cells within cultured tissue need simply to adapt to metabolic changes, depending on the culture media. These culture media are however detrimental as the media remain one of the main factors that need to be controlled closely to obtain reliable results.

We tested some different basic media but did not observe obvious changes regarding the cultivation of tumor slice cultures. But we assume that supplementary growth factors and media composition will have some impact on tissue integrity and survival of certain cell populations.

3D Spheroids Help Scientists Discover Smarter Cancer Drugs | Technology Networks

Although tissue slices were cultivated floating and not at a liquid-air interface, Naipal et al. Majumder et al. Moreover they observed a similar activation profile of some main receptor tyrosine kinases [18].

Individual serum, however also may carry concentrations of various therapeutic substances depending on individual medication. Therefore, it is also a matter of experimental question whether the media should be fully defined or supplemented with animal or even individual human serum. Defined culture media might even prolong the culture period of some tumor entities and enhance the survival of cellular populations within the tissue. This issue was already pointed out by Unger et al. However, nutritional uptake, oxygen supply and the influence of culture media on tumor slice cultures are still poorly explored.

Very few immunohistochemsitry IHC stainings demonstrate intact vessels in formalin-fixed, paraffin-embedded FFPE sections of tumor slice cultures [8]. However, Davis et al. These areas are observed in the tissue culture at the membrane-facing side. While this obvious limitation of the model urges for detailed investigation, the interesting point of tumor cells surviving hypoxic conditions might become a novel aspect of the tumor slice culture model. Still, the vasculature in tumor slice cultures needs to be closely investigated together with possible perfusion systems, not yet established in common tumor slice cultures.

These facts are detrimental, regarding drug delivery and uptake as most studies dissolve the drug of investigation in culture media. In sum we can say that even if diverse approaches are used in different studies, important considerations emerge and need to be addressed in future studies. More standardized research is needed to strengthen reproducible results and highlight the hallmarks of human tumor slice culture.

Human tissue is scarce and precious, therefore we carefully need to select experiments the tissue is most valuable for. The results were reported in the journal Oncogene. In contrast to single layers of cells normally used to screen for drugs, which tend to all grow at the same rate because they get the same exposure to oxygen and nutrients, the spheroids mimic what might happen in a tumor: Some cells are on the outside and some are on the inside.

In the new paper, the researchers focused on a cancer-driving protein called KRAS. They are common in lung cancer, colorectal cancer, and especially pancreatic cancer. In fact, up to 90 percent of pancreatic cancers are driven by KRAS mutations, and the investigators used pancreatic cancer cell lines for the current study. People have spent several decades trying, but so far there has been little success," says Joseph Kissil, PhD, professor at Scripps Research Medicine and the other co-corresponding author.

But the method of screening that we used in this study allowed us to come at the question in a different way. We were just looking for something that acted on some part of the pathway that's driving cell growth. The compound is similar to a class of drugs used to treat some heart conditions. Although the team says this particular drug is unlikely to be developed as a cancer treatment, it validates the approach of conducting drug screenings using spheroids.

In theory, you could use this screening method for any line of cancer cells, and any mutation you want. This study provides direct evidence that screening for drugs using 3-D structures of cancer cells may be more appropriate. Note: material may have been edited for length and content. Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biol ; Biotechnol J ; Lost in translation: Animal models and clinical trials in cancer treatment.

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Harnessing New Dimensions in Your Research: Coming ’Round to Cancer Spheroid Culture

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