Drug Toxicity

Determining toxicity is a major need and it is challenging.

Toxicity must be assessed using both short-term (single dose, 24–48h; acute) and long-term (multiple doses, weeks to months; chronic) culture conditions. This is because drug induced liver injury (DILI) can develop following a single acute exposure and from repeated chronic treatment (e.g. by development of drug tolerance or deposition of metabolites)​. Long term treatment is especially important because it corresponds to the usual treatment regimen for patients but is difficult to replicate in an in vitro system.

Animals have proven not to be an accurate model and 40–50% of the drug candidates associated with hepatotoxicity in humans did not present the same toxicological concerns in animal models.​

One of the reasons for this discrepancy is the differential expression and activity of drug metabolizing enzymes between animals and humans that might confound the extrapolation of data derived from model species.

 Ref. “A Critical Perspective on 3D Liver Models for Drug Metabolism and Toxicology Studies”, 2021, Serras et. al. Front. Cell Dev. Biol., 22, 9, 626805

Acute toxicity

The ClinoStar in vitro 3D spheroid system that can be used for determining toxicity.
It has been shown that using clinostat – based system as growth environment for immortal hepatoma cell line the model constructs responded correctly to treatment with six commonly used drugs. Use of immortal cell line makes the system very accessible, inexpensive and reproducible also between different laboratories. Cells grown in the system recover liver functionality to a level seen in in vivo tissue and some primary cells. Use of the system allows to select targets more accurately for further clinical studies and greatly reduce and improve use of laboratory animals

The drugs chosen were: amiodarone, diclofenac, metformin, phenformin and paracetamol [Fey et. al. 2020]  and valproic acid (VPA). The selection was based on their diversity of structure, target organ, biological half-life and cytochrome 450 enzyme that metabolizes them. Their important properties are presented in Table 1.

Table 1. Di, initial dose; Dm, maintenance dose.

An important question to be answered is how predictive the in vitro toxicity determination is compared with that observed in vivo (i.e. persons who accidentally or deliberately were exposed to lethal doses). This question was approached by averaging all the literature available for the selected. The data were divided into three categories:

• 2D cultures of HepG2– and HepG2–C3A
• Primary human hepatocytes
• 21 days old 3D cultures of HepG2–C3A.

All the in vitro toxicity data were converted to LD₅₀ (mg compound per mg protein) and compared with the in vivo comatose or lethal dose.

The answer was clear.

• HepG2 when grown in the classical 2D cell culture results in a poor correlation,  R² = 0.547.
• Fresh primary human hepatocytes (the pharmaceutical industry’s ‘gold’ standard) resulted in a better correlation,  R² = 0.747.
• HepG2–C3A 3D spheroids resulted in the best correlation, R² = 0.854.

Accumulated toxicity

In order to investigate the spheroids’ response to drug treatment in detail, spheroids were treated six times with various doses of paracetamol at 48-h intervals during a 10-day period . Samples were collected at the time the medium was exchanged and in addition, 3 and 6 h thereafter.

In total, 20 mg paracetamol per mg protein was shown to be acutely lethal. Half of this dose was found to be chronically lethal, where four treatments killed the spheroids.

Further 2-fold dose reductions stimulated ATP production. The initial fall in ATP levels was followed by a ‘defensive’ stimulation.

Interestingly, at the lower doses of 5, 2.5 1.25 and 0.625 mg/mg, the stimulation of the ATP response did not appear to be dose dependent. One possible explanation is that these cells have reached a maximum tolerable ATP limit.

The response to all doses became stronger after the first two treatments, suggesting that the spheroids were adapting. They may be producing additional glutathione and reducing rates of protein synthesis and degradation.

Both of these features are significant because multiple doses are relevant for patient treatment. The therapeutic dose is 4 g/day resulting in a blood concentration of 10–25 μg/ml. If the ATP levels are indicators of the therapeutic effect, then treatment is maximized already at very low doses. Thus, a lower therapeutic dose might be sufficient and maintenance doses could be even lower.

Conclusion

• HepG2/C3A spheroids can provide a practical model for the determination of lethal toxicity thresholds in vitro (both acute and chronic).
• Their ratio suggests that the average therapeutic index is 3-fold lower than commonly assumed. This might have significance for patients who repetitively take medicine.
• Some subtoxic drug concentrations activate ATP production. This is assumed to be a protective (or stress) response.
• Subtoxic drug treatment of spheroids results in a clear response and then a return to the ‘metabolic equilibrium’ from which the cells were displaced.
• Toxicity determinations made using C3A spheroids are at least as predictive as primary human hepatocytes of in vivo toxicity.

Finally, because of the simplicity of the ClinoStar system, it is possible to test repeated drug treatments over extended periods of time. The longest treatment presented here was six treatments in 10 days but could have been extended. Thus, HEPG2/C3A spheroids offer an inexpensive, highly reproducible alternative, which would be comparable between labs. Therefore, C3A spheroids can be used for repeated-dose screening of candidate drugs (whether natural compounds or synthetic).

CANCER Research

Cancer is a disease characterized by uncontrolled growth of abnormal cells. As its progression relies on growth and replication, it can spread to multiple organs. Cancer, also referred to as malignant tumors or neoplasms, is a leading cause of death worldwide, responsible for nearly 10 million deaths in 2020[i].

One defining feature of cancer is the creation of abnormal cells that grow beyond their usual boundaries, and which can then invade adjoining parts of the body and spread to other organs; the latter process is referred to as metastasis. Widespread metastases are the primary cause of death from cancer

The transformation of normal cells into malignant tumours is a multistep process, during which genetic mutations accumulate. Hanahan and Weinberg have described ten key changes that can occur during the transformation of a normal cell to a tumour cell; these features are considered hallmarks of cancer[ii].

Cell culture techniques

Most of the key techniques for cell culture were established during the 1950’s. The focus at that time was to make the cells proliferate as quickly as possible. This was done by allowing the cells to grow as monolayer cultures on essentially flat glass or plastic surfaces (i.e. in ‘2D’).

Validating cancer models

Most of the key techniques for cell culture were established during the 1950’s. The focus at that time was to make the cells proliferate as quickly as possible. This was done by allowing the cells to grow as monolayer cultures on essentially flat glass or plastic surfaces (i.e. in ‘2D’).

In order for a model to be valuable it has to successfully replicate in vivo conditions. Thus it has to meet  certain criteria to be validated.

These include:

• Growth rate
• Architecture and structure.
• Function
• Uniformity
• Reproducibility
• Longevity
• Biomarkers

1) Growth Rate

Cells in 2D cultures usually can double their numbers within a few days. Cells in normal tissues, cancers and in 3D cultures usually double their numbers in months.

2) Architecture and structure

Cells grown in 2D show no tissue architecture whereas the same cells grown in 3D can spontaneously form structures which resemble their parental tissue[i]. Key to this is that spheroids develop oxygen, nutrient and waste product gradients similar to those seen in tumours.

3) Function

In order for a 3D cancer model to be relevant for toxicology studies, it must successfully replicate the functionality demonstrated by the active tumour.

4) Uniformity

In order for a 3D cancer model to be relevant for toxicology studies, it must successfully replicate the functionality demonstrated by the active tumour.

5) Reproducibility

Reproducibility is inversely proportional to heterogeneity. Patients are heterogeneous. Their tumours are heterogeneous. The cells within a tumour are heterogeneous. It therefore makes no sense to increase this heterogeneity even further by growing the cells in conditions which force them to adapt. 

6) Longevity

Once cells have repaired the damage, they enter into a ‘dynamic equilibrium’ state: a state that resembles a functioning organ. Unperturbed, the cells execute their functions at a steady rate. Treated with a drug (or other biologically active molecules) they respond and when the drug is metabolised (or removed) the cell clusters return to their original dynamic equilibrium. In the Clinoreactors, cell clusters can remain in this dynamic equilibrium for weeks or months and this is an ideal starting point for experimentation.

7) Biomarkers found using 3D culture

Efficient and timely identification of malignant tumours forms the basis of cancer treatment. Recently, accurately detecting cancer-specific biomarkers have become the pathologist’s chosen tool to study biopsies. In order to build this toolkit with biomarkers specific to the myriad of cancer types, it is necessary to conduct gene expression profiling of the appropriate tissue. One way to do this is to employ 3D culture to study tumour extracts and cancer cell lines.

Cancer and ClinoStar system

When it comes to replicating in-vivo standards, it is clear that 3D culture is far superior to 2D. To generate 3D constructs which will mostly closely mimic in-vivo conditions, it is necessary to culture them in an environment that is an accurate biological representation. The ClinoStar system is a 3D bioreactor platform which employs the clinostat principle to grow spheroids. It provides a ‘microgravity’ simulated environment which produces highly reproducible  3D cultures. The system  allows cultures to be grown and matured over longer periods of time, allowing investigation of mimetic tissues to occur in a manner comparable to tissues in the intact organism.

Cell line	Cancer type	Used for	Conclusion	Picture
H69V	Small cell lung cancer	model for anticancer treatment screening	It can be concluded that the established functional NCI-H69V spheroid model is viable for at least 30 days, and can be used for experiments from 14 days of culture in the bioreactor
LS180	Colorectal	model for anticancer treatment screening	LS180 sodium alginate encapsulated spheroid model could be used for testing new chemotherapeutic compounds for colorectal cancer.
FTC-133	follicular thyroid carcinoma		Developing a model for understanding thyroid cancer

[i] Lelièvre, S.A., Kwok, T, Chitiboyina, S. Toxicol In Vitro 2017, 45(Pt 2); 287-295

^vii Three-dimensional cell culture: A powerful tool in tumor research and drug discovery – PMC (nih.gov)

^viiiDesRochers TM, Suter L, Roth A, Kaplan DL. Bioengineered 3D Human Kidney Tissue, a Platform for the Determination of Nephrotoxicity. PLoS ONE. 2013; 8:e59219. [PubMed: 23516613]

^ixOhmori T, Yang JL, Price JO, Arteaga CL. Blockade of tumour cell transforming growth factor-betas enhances cell cycle progression and sensitizes human breast carcinoma c

^x Jayanta DJS, Brugge. 2005. Modelling glandular epithelial cancers in three dimensional cultures. Nat. Rev. Cancer. (5):675-88.

^xi Paraic AK, Genee YL, Connie AM, Richard MN, Jeremy RS, Paul TS. 2007. The morphologies of breast cancer cell lines in three-dimensional assays correlate with their profiles of gene expression. Mol. Oncol. 1:84-96

^xii Kumar HR, Zhong X, Hoelz DJ, Rescorla FJ, Hickey RJ, Malkas LH, et al. 2008. Threedimensional neuroblastoma cell culture: proteomic analysis between monolayer and multicellular tumour spheroids. Pediatr. Surg. Int.24:1229-34

^xiii  Li GN, Livi LL, Gourd CM, Deweerd ES, Hoffman-Kim D. 2007. Genomic and morphological changes of neuroblastoma cells in response to three-dimensional matrices. Tissue Eng. 5:1035-47.

Our technology has been used for cultivating:

• Human adenocarcinoma cell line: HeLa
• Human small cell lung cancer: NCI-H69 and sub clones
• Rat insulinoma cell line: INS-1 and INS-1E
• Rat islet cell line: RIN-m5F
• Mouse pre-adipose embryo fibroblast cell line: 3T3-L1
• Freshly isolated monkey (baboon) and rat islets of Langerhans
• Freshly isolated human colon cancer biopsies