Advantages and disadvantages of
Abstract
Cancer represents the leading cause of global mortality worldwide. Recent estimates have shown that approximately 25% of all cancer types exhibiting KRAS mutations, making these mutations one of the most reported so far. Given the important role played by KRas during the progression of different tumors, the search for new therapeutic compounds that can reduce the adverse effects of this oncogene becomes evident. However, discovering effective anticancer compounds is a complex and time-consuming task. These compounds should ideally exhibit potent anticancer properties at low concentrations, with minimal impact on healthy cells. The validation of potential candidates involves several stages and methods, including in vitro techniques such as cell lines or primary cell cultures grown under 2D and 3D conditions. This chapter provides a comprehensive review of in vitro methods to support the effectiveness of two compounds, C14 and P8, specifically targeting mutant KRas as potential antitumor agents. Cytotoxicity assays were employed on breast and pancreatic cancer cell lines and primary cell cultures grown in 2D and 3D conditions to evaluate the effectiveness of these compounds. The use of multiple cell culture systems provides more pertinent data, enhancing our understanding and assessment of the potential benefits of new therapeutic molecules.
Keywords
- human primary cell
- 3D cell cultures
- K-Ras4B
- PDE6δ
- therapy
1. Introduction
Cancer is the leading cause of death worldwide. According to GLOBOCAN, every year, over 20 million people are diagnosed with cancer globally, which leads to nearly 10 million deaths reported in 2020 [1]. The increasing incidences of cancer worldwide have created a demand for identifying and developing effective and tolerable therapies for treating different types of cancer [2].
Among the most common causes of cancer death in 2020 were: lung, colorectal, stomach, breast and pancreatic cancer [1]. It is important to highlight that, from these six pathologies, they share in common the presence and dependence of
However, its function has been shown to be frequently lost during tumor progression in many types of cancer. The disruption of KRAS regulation is triggered by specific point mutations occurring at codons 12, 13, 61, and 146 within the
For decades, several groups made unsuccessful attempts to target K-Ras in cancer treatment due to the protein’s unique characteristics, specifically the absence of binding grooves on its surface for small molecules. As a result, K-Ras was considered undruggable [12, 13, 14]. However, in 2013, an innovative strategy emerged with the discovery that PDE6δ plays a role in regulating the subcellular location of K-Ras. In this research, inhibiting the interaction between both proteins resulted in K-Ras being retained in the cytoplasm, which impacted its ability to transmit signals. Consequently, the K-Ras-PDE6𝛿 complex was identified as a potential pharmacological target [15]. However, these strategies have shown be limited to a single K-Ras mutant, and toxic to non-cancerous cells due to their mechanism affecting both K-Ras WT and other proteins recognized by PDE6𝛿 [15, 16].
In 2018, our research group presented a new strategy. We proposed the stabilization of the K-Ras/PDE6𝛿 complex by small molecules instead of preventing its interaction (Figure 1). Based on this, a series of compounds capable of binding and firmly fixing the interaction between the K-Ras and PDE6𝛿 proteins were selected
The antitumoral efficacy of these molecules has been assessed in various
Given the high prevalence of cancer, there is a need to find novel drugs to treat this complex disease. These new treatments should have the minimal side effects on the patients, while also being more effective in eliminating malignant cells and showing low rates of resistance to them. For these reasons, it is necessary to evaluate new therapeutic options in many models to achieve their validation.
This chapter reviews the most common
2. In vitro models for anti-cancer drug discovery
The process of drug development and its evaluation can indeed be costly. As a result,
Cell lines | Primary cultures | 3D models | |
---|---|---|---|
Accessibility | Can be purchased from a company (derived from established cell lines) | Derived directly from isolated tissues. | Significant time must be invested in developing these models (generated from cell line or primary culture cells) |
Proliferation | Unlimited | Limited | Limited |
Characterization | Well characterized | Must be characterized | Must be characterized |
Complexity and genetic variation | Simpler, with fewer cell types. Minimal genetic variation. | Presents higher heterogeneity, with various cell types. High genetic variation that reflects the genetic makeup of the tissue of origin. | Higher complexity than 2D cultures. Resembles tumor structure. Higher genetic variation. |
Biological relevance | May not fully represent | Closer to | Closer to 3D cell culture models more closely mimic Additionally, protein and gene expression levels in culture resembling those found |
Ease of result interpretation | Results are easier to interpret | More challenging to interpret due to a higher number of variables involved | More challenging to interpret due to a higher number of variables involved |
Ease of handling | Uncomplicated to handle | More challenging to handle | More challenging to handle |
Cost | Less expensive to maintain | Cost is higher at the beginning when the cell line is being established. | More costly to maintain. |
2.1 2D cell culture for anticancer drug testing
2D cultures are the most employed cell models to test drugs due to two major advantages: the methods to cultivate and preserve the cells under these conditions are uncomplicated and importantly; maintenance in terms of materials that are needed is low-cost and being a simpler model interpretation of results is easier [24].
In this classic model, cells grow as a monolayer in a polystyrene culture flask or petri dish [24]. By definition, a cell line is a collection of cells originated from one cell. These cell lines are
As previously stated, our research group has evaluated the antineoplastic effects of a group of compounds capable of stabilizing the K-Ras-PDE6𝛿 complex. To validate their effects in 2D conditions, we utilized various pancreatic and breast cancer cell lines (Table 2). Additionally, antitumoral effects were determinate by viability assays were used.
Pancreatic cancer-derived cell lines |
hTERT-HPNE (ATCC, CRL-4023). Cultured in 75% DMEM without glucose (Biowest) with additional 2 mM L-glutamine (Gibco) and 1.5 g/L sodium bicarbonate plus, 25% Medium M3 Base (Gibco). The media is supplemented with fetal bovine serum 5%, 10 ng/ml human recombinant EGF (Sigma-Aldrich), 5.5 mM D-glucose (1 g/L) (Sigma-Aldrich) and 750 ng/ml puromycin (Sigma-Aldrich) at 37°C in an atmosphere with 5% CO2. |
BxPC-3 (ATCC, CRL-1687). Cultured in RPMI medium (Biowest, USA) supplemented with 10% fetal bovine serum and 1% antibiotic 100 U/mL (penicillin/streptomycin) and 1.5 g/L sodium bicarbonate at 37°C in an atmosphere with 5% CO2. |
Panc-1 (ATCC, CRL-1469). Cultured in DMEM medium (Biowest, USA) supplemented with 10% fetal bovine serum and 1% antibiotic 100 U/mL (penicillin/streptomycin) and 1.5 g/L sodium bicarbonate at 37°C in an atmosphere with 5% CO2. |
MIA PaCa-2 (ATCC, CRL-1420). Cultured in DMEM medium (Biowest, USA) supplemented with 10% fetal bovine serum and 1% antibiotic 100 U/mL (penicillin/streptomycin) and 1.5 g/L sodium bicarbonate at 37°C in an atmosphere with 5% CO2. |
Capan-1 (ATCC, HTB-79). Cultured in Iscove’s Modified Dulbecco’s Medium (ATCC) supplemented with 20% fetal bovine at 37°C in an atmosphere with 5% CO2. |
Breast cancer-derived cell lines |
MCF 10A (ATCC, CRL-10317): Cultured in Dulbecco’s modified Eagle’s medium F12 (DMEM F12) (Gibco, New York, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotic 100 U/mL (penicillin/streptomycin) (Sigma-Aldrich) and 1.5 g/L sodium bicarbonate (Sigma-Aldrich) at 37°C in an atmosphere with 5% CO2. |
MDA-MB-231 (ATCC, HTC-26): Cultured in Lebovitz (L-15) medium (Gibco) supplemented with 10% fetal bovine serum and 1% antibiotic 100 U/mL (penicillin/streptomycin) and 1.5 g/L sodium bicarbonate at 37°C in an atmosphere with 5% CO2. |
MDA-MB-231 RR (ATCC, HTC-26): Cultured in DMEM F12 medium (Gibco) supplemented with 10% fetal bovine serum and 1% antibiotic 100 U/mL (penicillin/streptomycin) and 1.5 g/L sodium bicarbonate at 37°C in an atmosphere with 5% CO2. |
MCF 7 (ATCC, HTB-22): Cultured in DMEM medium (Biowest, USA) supplemented with 10% fetal bovine serum and 1% antibiotic 100 U/mL (penicillin/streptomycin) and 1.5 g/L sodium bicarbonate at 37°C in an atmosphere with 5% CO2. |
MCF 7 RR (ATCC, HTB-22): Cultured in DMEM F12 medium (Biowest, USA) supplemented with 10% fetal bovine serum and 1% antibiotic 100 U/mL (penicillin/streptomycin) and 1.5 g/L sodium bicarbonate at 37°C in an atmosphere with 5% CO2. |
2.1.1 Pancreatic cancer cell lines
About 90% of pancreatic cancers are histologically classified as pancreatic ductal adenocarcinoma (PDAC) [26], and although it ranks as the 12th most common cancer, for decades it has firmly held the first position as the cancer type with the lowest 5-year survival rate [27]. Although a general mutational profile has been well determined for PDAC, the standardly gene reported as mutated is
2.1.2 Breast cancer cell lines
According to Globocan, breast cancer (BC) ranks first in incidence and second in mortality among all oncologic malignancies worldwide [1]. This is a heterogeneous group of diseases that can be grouped into four subtypes into four subtypes (luminal A, luminal B, HER2 and Triple negative Breast Cancer (TNBC)) [31, 32]. Regarding the genomic landscape of BC, various alterations have been identified within each subtype. Among them, mutations in
For initial drug screening it is suggested to determine the proper concentration to use in the desired assays through dose-esponse curve and IC50 determination. This process is often performed in 2D assay of the cell line of interest. The cells are plated and treated with various concentrations of the test compounds, typically in an eight-point dose range, along with vehicle and media only controls The methods are described in detail in Ref. [39].
2.2 Methods for viability/cytotoxicity methods determination
Cell viability or cytotoxicity in monolayer cultures can be determined by different methods. Cell viability assays are frequently employed, with a variety of markers as indicators of metabolically active (living) cells, such as measuring ATP levels (e.g., CellTiter-Glo® Luminescent assay), measuring the ability to reduce a substrate (e.g., tetrazolium reduction and resazurin reduction), and detecting enzymatic/protease (e.g., CellTiter-Fluor assay) activities present only in living cells.
The most widely used technique is Tetrazolium Reduction Cell Viability Assays, which measures cellular metabolism as an indicator of cell viability, proliferation, and cytotoxicity. In this method, the positively charged dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), penetrates viable cells and is metabolized into a purple-colored product known as formazan. As a result, color formation can be a helpful indicator of viable cells. However, the incubation time for this method is long (more than 4 hours). To address this limitation, other negatively charged compounds such as (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) (XTT), which cannot penetrate cells, must be combined with intermediate electron coupling reagents that help them enter into the viable cells. Once inside the cells, XTT is reduced to tetrazolium, a soluble formazan product. The incubation time for this method is 1–4 hours, making it more convenient [40]. For our experiments, we used MTT and XTT assays to build a dose-response curves and determine the effect of different concentrations of compounds C14 and P8 in pancreatic and breast cancer cell lines viability.
Anti-cancer drugs C14 and P8 should demonstrate inhibition of cancer cells growth or proliferation, and these is most often determined by IC50. In this work, IC50 is defined as the particular inhibitory drug concentration required to reduce the percentage of viable cells by 50% compared to cells grown without drug exposure [41].
The IC50 value (molar concentration) is important for
2.3 Evaluation of compound targeting K-Ras4B/PDE6δ in pancreatic cancer cell lines
A dose-response curve to evaluate the antitumoral effects of compounds of interest was performed using MTT assays. Pancreatic cancer cells were plated in 96-well plate and treated with various concentrations of C14 and P8 compounds, along with vehicle (DMSO) and media-only as controls. The IC50 concentration was determined using Prism 8 software (GraphPad, USA).
As observed below, C14 possess a high cytotoxic property in eliminating cancer cells (Figure 5B–E, blue line, and Figure 5F). On the other hand, P8, an analogue of C14, demonstrates a more potent cytotoxic effect on cancer cells (Figure 5B–E, red line, and Figure 5F) compared to the vehicle (Figure 5B–E black line).
Another crucial point is the specific effect of C14 and P8 over malignant cells. As the results showed above,
Remarkably no inhibitory effect was observed in the viability of the control cell line hTERT-HPNE, even at concentrations higher than 200 μM after 48 hours of exposure (Figure 5A, red and blue line and Figure 5F).
These IC50 values of C14 and P8 in cancer cell lines are lower than 90 μM, which is 2 and 6 times less than the concentrations required to affect the viability of control cells (Figure 5A and F). This suggests that these compounds could be an excellent treatment alternative for pancreatic cancer, as they exhibit cytotoxicity to cancer cells while sparing normal pancreatic cells from side effects.
As a result, this assay allows to observe the specific cytotoxic effect of compounds on the viability mainly in
2.4 Evaluation of compound targeting K-Ras4B/PDE6δ in breast cancer cell lines
As mentioned earlier, dose-response curves were generated to determine the cytotoxic effect of compounds C14 and P8 on the breast cancer cell lines using XTT assays. The breast cancer cells were plated in 96-well plate and treated with various concentrations of the compounds, with DMSO used as a vehicle and media only as control. The IC50 concentration was determined using Prism 8 software (GraphPad, USA). These data are presents in Figure 6.
Similar to observed in pancreatic cancer cells, no noticeable effect on the viability of the control cell line MCF 10A was observed after its exposure to C14 and P8 (Figure 6A, lines red and blue and Figure 6F). However, both compounds exhibited a potent cytotoxic effect over the rest of the tested breast cancer cell lines tested (Figure 6B–E, lines red and blue and Figure 6F). According with the IC50 values, even at concentrations higher than 100 μM after 72 hours of exposure, both compounds did not have impact over the viability of non-tumoral cells (Figure 6A and F). This finding supports the notion that C14 and P8 have a specific effect over
Taken together, these findings indicate that the conventional 2D cell-based cancer drug screening on pancreatic and breast cancer cell yields reproducible results of the specific cytotoxic effect over cancer cells without affecting non-tumor control cells. This makes them suitable for further experiment and for evaluating their effects in a more complex cell culture.
Primary cultures generated directly from tumors are a viable alternative to employing cell lines. In comparison to cell lines, in which all cells are genetically identical, primary culture cells gene expression can vary from cell to cell. Primary cultures offer several advantages. Not only are cells isolated directly from the tumor site but provide the complete pathology which allows to compare the properties of the culture to those of the original. In general, such cultures can be generated as explants, in which mixed cell populations develop out of small fragments of tissue, or as enriched populations of particular cell types, such as cancer cell or cancer stem cells, being ideal to evaluate the efficacy of a new anticancer drug [44].
3. Primary cell culture for anticancer drug testing
Primary cultures consist of cell cultures derived directly from isolated tissue cells (Figure 7). These cultures can proliferate under 2D and 3D conditions without undergoing any genetic modifications. However, the number of passages for their use is highly limited [45]. Primary cultures derived from malignant tumors are particularly of great interest in research. Various protocols are currently available for obtaining primary cell cultures, and the selection of a specific method depends on factors such as tissue type and sample size. In Table 3, summarizes the different methods for obtaining primary cell cultures in 2D from pancreatic cancer tumor samples [46, 47, 48]. Once the primary cultures are obtained, they must be characterized to determine the presence of the required cell lineage. Currently, various methods facilitate their characterization including, the evaluation of specific markers by immunofluorescence microscopy or Fluorescent Activated Cell Sorter (FACS) (e.g., Figures 8 and 9, respectively). The most commonly used antibodies for malignancy markers are: E-Cadherin, Ki-67, Vimentin, B-Catenin, MUC-1, MUC-4, MUC-16, EGFR, CEA, lineage markers: CK19 and CK7, and for cancer stem cells (CSCs) marker: CD24, CD44 and CD133 [8, 49, 50, 51, 52].
Method | Mechanism | Advantages | Disadvantages |
---|---|---|---|
Explant | The tissue is cut into several fragments with the aid of a scalpel or dissecting scissors. | The cells coming from the niche leave and give rise to the exit of the tumors | Culture is very slow as there is no cell dissociation |
Chemical | A tissue fragment is placed in a 1.5 mL tube and 10 volumes of 0.5% EDTA are added. | Intercellular bonds are broken. | Cells do not dissociate. |
Mechanic | Cut, scratch, the tissues into small pieces to separate the cells and wash them with gentle agitation. | It is a fast technique for soft tissues. | Cells are not dissociated, and cell viability may be decreased. |
Enzymatic | Enzymes for cutting or digesting pieces of tissue into free cells, collagenase and trypsin are commonly used. | Cells dissociate more easily. | Enzymatic cleavage can modify proteins on the surface of cells. |
Combination | Combination of the 4 previous methods. | A greater number of cells are obtained, and a suitable niche can be generated. | Caution with the times. |
3.1 Isolation and characterization of primary cultures derived from pancreatic cancer
Primary PDAC cultures were obtain in the laboratory using an enzymatic method involving collagenase and trypsin (1:5 trypsin/EDTA solution, 15 min until the tissue was loose) under sterile conditions (Table 3 and Figure 7). The requirements to maintain the primary culture vary depending on the aggressiveness of the cancer and the stage of the cancer being studied. The percentage of serum and growth factors is gradually decreased until the cells can survive in standard conditions (10% serum and 1% antibiotic) (Table 4).
Requirements | P0 | P1–3 | P4–10 |
---|---|---|---|
Medium | DMEM High Glucose or Advance DMEM F12 Medium | DMEM High Glucose or Advance DMEM F12 Medium | DMEM High Glucose or Advance DMEM F12 Medium |
FBS | 70% | 50% | 10% |
Matrigel | 0.1% | — | — |
Antibiotic/antimycotic | 5% | 3% | 1% |
EGF | 10 ng/mL | 10 ng/mL | — |
Other | Ciprofloxacin 10ug/mL | Ciprofloxacin 10ug/mL | — |
The characterization of those cultures previously was previously performed using immunofluorescence microscopy employing the specific markers, CD44+ (Ambion, USA) (Figure 8A), CD24+ (Ambion) (Figure 8B) and CD133+ (Ambion)(Figure 8C). These assays strongly suggested the presence of cancer stem cells (CSCs), in both cultures. To verify this information, flow cytometry assays were conducted which revealed the presence of CSC in 2.4% of total population of MGKRAS004 cells and in 2.18% of MGKRAS005 population (Figure 9A).
Based on these results, there is a high abundance of CSCs in both primary cultures. This finding is significant due CSCs have the ability to contribute to the development of a more aggressive tumor characterized by high rates of proliferation, invasion, and resistance to conventional therapies [53]. Keeping this in mind, C14 and P8 compounds were evaluated on these primary cell cultures.
3.2 Evaluation of compounds targeting K-Ras4B/PDE6δ on primary cultures from pancreatic tumors
After demonstrating the heterogenicity and complexity of pancreatic primary cultures, the efficacy of C14 and P8 compounds was evaluated on 2D conditions using primary cultures obtained from samples of pancreatic malignant tumors, with the same conditions used for cell lines and DMSO as control (Figure 10A and B). According to our evaluation, both molecules were more effective against the MGKRAS004 cell line. In this case, C14 exhibited an IC50 of 38.72 μM, while P8 showed an IC50 of 80.88 μM with (Figure 10A and C). These IC50 values are consistent with those obtained for the common pancreatic cell lines (Figure 5F). However, in the case of MGKRAS005, the effect of C14 was reduced, and an IC50 of 268.2 μM was observed (Figure 10B and C). A similar trend was observed with P8, where the IC50 observed was 148.9 μM (Figure 10B and C). The explanation for this data could be related to the amount of CSC present in each population. As mentioned earlier, both primary cultures contain a similar proportion of CSC. However, in the case of MGKRAS005, the population of cells expressing CD133 is greater (Figure 8C). This higher expression of CD133 in MGKRAS005 cells may contribute to their reduced sensitivity to C14 and P8, leading to higher IC50 values observed in the treatment response. These data are in agreement with previously reports that associated this molecule with chemoresistance in various cancer cells [54].
It is important to highlight that many drug candidates will fail during clinical trials, leading to the loss of money and time invested in research.
Although the most used cell culture growth conditions in cancer research are 2D models, they do not accurately reflect the native environment and general physiology of malignant cells and tumors such as, mechanical and biochemical signaling or intercellular communications [55], cell invasion [56] and expression of pathological markers [56]. To address these limitations, three-dimensional (3D)
4. 3D models for anticancer drug testing
Currently, various types of 3D models are available for cancer research. The most commonly used ones include spheroid models, organ-on-a-chip models, hydrogel models, and bio-printed models [57]. Each of these approaches has its own specific advantages and disadvantages. However, despite the significant time and cost involved in their production, spheroids are the most widely utilized due to their unique characteristics [60].
Spheroids are small-scale 3D models that could self-assemble into spherical cell aggregates (with radii ranging from 100 to 600 μm) [60, 61, 62]. These models exhibit various tumoral characteristics, such as, a central necrotic core, surrounded by quiescent cells and an outer layer consisting of actively proliferating cells [60]. Other important features observed are, pH, oxygen, and metabolic compound gradients [63], and in some cases, micrometastases [64].
In this section, the focus is on spheroids, generated from cell lines or cells obtained from primary cultures of tumoral tissues. The following will describe the procedure for creating spheroids using cell lines (refer to Table 2) as well as spheroids from primary cultures, along with the method for characterizing their cellular composition. Subsequently, the cytotoxic effects of compounds C14 and P8 on the 3D models will be discussed.
4.1 Spheroids derived from cell lines and primary cell cultures
The following method outlines the protocol utilized by our research group to generate spheroids from cell lines or primary cultures (Figure 11). Firstly, the process begins with expanding of cultures under 2D conditions. Cell harvesting is conducted using a standard procedure. Briefly, when the cells reach 70-80% confluency in a plate, the medium is removed, and the cells are collected using trypsinization (0.05% trypsin, 0.53 mM EDTA, Gibco, USA). Subsequently, the cells are centrifuged and resuspended in 1–5 ml of spheroid medium, consisting of DMEM/F12 (Gibco), 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin (Sigma-Aldrich). The spheroid medium is further supplemented with 20 ng/ml recombinant human epidermal growth factor (Sigma-Aldrich), 10 ng/ml recombinant human basic fibroblast growth factor (R&D Systems, USA), and 1X B27 supplement (Sigma-Aldrich).
From this cellular suspension, approximately 3000–5000 cells are seeded per well in 96-well ultra-low adherence plates (Gibco). The plates are then maintained at 37°C and 5% CO2 for a period of 4 to 7 days, depending on the cell line and desired spheroid size. During the initial 5-day growth phase, it is crucial to minimize disturbances to the plates, particularly during the initial 5-day growth phase [65]. At the end of the culture period, spheroids are collected through gentle centrifugation at 800 rpm and utilized in functional assays.
Alternatively, enzymatic dissociation of the 3D models can be achieved by incubating the culture for 10 minutes with 0.05% trypsin (Invitrogen), followed by mechanical pipetting using a flame-polished Pasteur pipette. The dissociated cells are then passed through a 40 μm pore size filter (Corning). Subsequently, the cells can be analyzed for specific molecule expression using techniques such as FACS [66] or conventional immunofluorescence microscopy.
Figure 11 shows all the cell lines and cells from primary cell culture, that were able to develop spheroids. In the case of MDA-MB-231, this cell line is able to generate spheroids in ultra-low adherence plates, although they present a less compact appearance and less well-defined edges (Figure 11) [61]. Something similar is observed in its derivative radioresistant cell line (Figure 11). On the other hand, MCF 7 and MCF 7RR cells, quickly formed created three-dimensional spheroids with a homogenous inner structure when placed on plates with extremely low attachment properties as previously shown [67, 68]. Finally, although the structure of 3D models derived from primary cell cultures MGKRAS004 and MGKRAS005 is looser and more challenging to obtain compared to cell lines, these models provide a closer representation of the
4.2 Calculation of spheres formation efficiency (%)
To verify the effectiveness of the aforementioned method, the percentage of spheroids can be determined. This involves counting the number of spheroids (larger than 40 μm) after the culture period using a microscope at 40X magnification. Digital images of 5 random fields are captured using a digital camera connected to an optical microscope, and the size of the spheroids is determined using acquisition software. The spheroid formation efficiency (MFE%) is calculated using Eq. (1).
4.3 Fluorescent activated cell sorter (FACS)
As mentioned earlier, the characterization of spheroids involves various methods, including FACS. According to previous reports [70, 71], the generation of spheroids leads to the enrichment of a subpopulation known as Cancer Stem Cell (CSC). These CSCs population, are cell that are distinguished by their high tumorigenic potential, which includes self-renewal, pluripotency, and proliferative abilities. Additionally, they exhibit resistance to conventional treatments such as chemotherapy and radiation [72, 73]. Given these characteristics, CSCs are widely used as therapeutic targets for new drugs.
The following section outlines, the method employed by our research group to evaluate the expression of specific CSC markers using FACS in spheroids. This approach is employed to determine the antitumoral effect of the compounds of our interest. The cells derived from the spheroids are dissociated, as described above, and then stained with the following antibodies: anti-CD24-phycoerythrin (PE) (Abcam, USA), anti-CD44-allophycocyanin (APC) (Abcam), and anti-ALDH1-fluorescein (FITC) (Abcam). The cellular staining is conducted according to a procedure previously reported [66]. In brief, the dissociated cells are passed through a 40 μm cell filter (Corning) and counted to adjust the concentration to 100,000 cells/ml. Each antibody is diluted at (1:100), added and incubated at 37°C for 1 hour. After incubation, the cells are washed three times with PBS. Subsequently, flow cytometry analysis is performed.
4.4 Enrichment of CSC in spheroids derived from primary cell cultures
In the following section, representative data of cells derived from primary cultures of pancreatic tumors are presented. FACS assays demonstrated the presence of CD44+, CD24+, and CD133+ markers in spheroids derived from primary cultures (Figure 9) [74]. These findings support the existence of CSCs in spheroids obtained from MGKRAS004 and MGKRAS005 samples [75]. As mentioned earlier, the generation of spheroids selectively enriches the CSC population. The enrichment of CD44+, CD24+, and CD133+ marker expressions in spheroids, compared to 2D cultures, further confirms the selective effect of 3D systems on this cell population (Figure 9C). In the case of MGKRAS004, 2.4% of CSCs were found in 2D cultures, whereas this percentage increased to 6.3% in 3D conditions (Figure 8A and B). Similar results were observed for MGKRAS005 cells, with 2.18% of CSCs detected in 2D cultures and a significant increase to 24% in 3D cultures (Figure 9A and B). These findings also support the utilization of these 3D models as a more realistic system to evaluate the effects of new therapeutic compounds. It is worth noting that the levels of CD44+ and CD24+ in patients have recently been associated as predictors of mortality [76].
4.5 3D cell viability assay and IC50 determination
Using the 3D models described above, the cell viability effects of compounds C14 and P8 were determined. For this purpose, the CellTiter-Glo® 3D Cell Viability Assay Reagent from Promega (USA) was employed. This method measures ATP as an indicator of viability and provides a luminescent readout. Briefly, spheroids were generated as mentioned previously, and then exposed to both compounds for 48 hours. Cell lysis and luminescent signal recording were performed according to the manufacturer’s instructions, and IC50 values were calculated using Prism 8 software (GraphPad, USA).
In the 3D models, although C14 and P8 reduced the cell viability of spheroids, their IC50 values were higher compared to the 2D conditions. For MGKRAS004, the IC50 for C14 in 3D was 395.7 μM, whereas it was 38.72 μM in 2D (Figure 12A, C and Figure 10A, C). P8 had an IC50 104.4 μM in 3D and 80.88 μM in 2D (Figure 9A). In the case of MGKRAS005, C14 exhibited an IC50 of 470.4 μM in 3D and 268.2 μM in 2D (Figure 12B, C and Figure 10B, C). On the other hand, P8 showed an IC50 of 96.71 μM in 3D and 148.9 μM in 2D (Figure 12B, C and Figure 10B, C). This increase in IC50 values in 3D models is a phenomenon that has been previously demonstrated [59] and is attributed to the greater complexity observed in these systems. In conclusion, despite the increased IC50 values observed 3D culture conditions, both compounds remain active. In this regard, it is evident that compound P8 exhibited better activity, which aligns with its chemical attributes as a compound selected as a C14 analog with enhanced activity. Thus, P8 demonstrates antitumor properties on CSC-enriched populations.
5. Drug screening: Challenges and troubleshooting
The search for new antitumor compounds is a long and challenging process. Demonstrating the effectiveness of candidate compounds must go through different stages. Firstly, they should be highly effective in exerting their effects at low concentrations, while also desirably minimizing their adverse effects on non-tumor targets.
Ensuring the effectiveness of novel antineoplastic candidates requires extensive evaluations in both
Another crucial aspect that we deem essential during the implementation of the various methods presented in this chapter is their strict standardization to ensure the precision obtained results. This becomes particularly important when working with complex models like 3D cultures. Based on our own experience, incorporating procedures that enhance the homogeneity of mammospheres, such as gently centrifuging cells during seeding, has proven to be highly advantageous.
Finally, we emphasize the significant importance of developing methodologies to assess the physiology of 3D models. Although several effective systems exist for evaluating different aspects in 2D conditions, corresponding tools for 3D cultures are often scarce or nonexistent. Therefore, research groups must take the initiative to develop these methods themselves.
6. Conclusion
2D models are the standard for drug evaluation keeping the cost lower and requiring fewer cells and less time compared to animal models or human clinical trials. However, this model does not closely mimic the behavior and drug metabolism seen in
For more reliable inhibitory effects and better characterization of drugs, primary and 3D models are preferable for analyzing pathologies with the highest mortality, such as cancer. Primary cells provide broader spectrum of cell types from a greater number of patients to be studied without inducing artificial genetic mutations, and maintaining the same phenotype throughout the culture, while keeping the advantages of a 2D cell line-base assay in terms of time and cost. Nevertheless, management of primary cell culture poses challenges. Obtaining the patient consent, quality of samples, practicing sterility, and maintaining the culture are some of the challenges faced when working with primary cultures. To overcome these difficulties, proper collection with the help of pathologist and selection of appropriate isolation methods and culture media based on tissue type can help to increase the cell viability.
On the other hand, 3D cell culture models offer a number of advantages as they better represent the microenvironment of
In conclusion, this chapter reviews several
Acknowledgments
Dayan Andrea Carrion Estrada and Sandra Delfin Azuara received a pre-doctoral scholarship from CONACyT (935796 and 1078870). Dr. Paola Briseño Díaz, researcher, thanks to the Faculty of Medicine of UNAM and CONAHCYT for their support in carrying out this work. We thank Dr. Maria del Rocio Thompson Bonilla from the ISSSTE Mexico Hospital 1° de Octubre, since without her support the primary cultures would not have been performed. We thank Dr. Elena Arechega from the UAM Cuajimalpa Mexico, for the donation of radioresistant breast cancer cell lines.
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