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 KRAS gene mutations [3].

KRAS (Kirsten rat sarcoma viral oncogene homolog) was first reported to be a transforming gene related to oncogenesis in humans in the early 1980’s [4, 5]. Since then, KRAS has emerged as one of the extensively studied proto-oncogenes, owing to its established mutation prevalence of approximately 90% in pancreatic adenocarcinomas, 45% of colorectal adenocarcinomas, 22% of lung adenocarcinomas and 5% of breast cancers [6]. KRAS mutations are found in 30% of all cancers, making it the most frequently mutated oncogene across all cancer types and a promising drug target due to its participation in the progression and maintenance of several types of tumors [7].

KRAS belongs to the RAS family of small GTPases and behaves as a molecular switch by cycling between a GTP-bound active state and a GDP-bound inactive state. Wild-type KRAS is a tumor suppressor that is usually activated via specific receptors to regulate different signaling pathways involved in, cell cycle progression, cell growth, proliferation, migration, and apoptosis (Figure 1). For K-Ras protein to be active it needs to be transported to the cell membrane, through phosphodiesterase 6 delta protein (PDE6δ) [10, 11]. The interaction between K-Ras and PDE6δ is performed by the recognition of a post-translational modification (farnesyl group) present in the GTPase (Figure 1).

Figure 1.
RAS effector pathways. Plasma membrane-associated Ras-GTP can directly interact with multiple different effectors to activate different signaling pathways such as cell proliferation, migration, survival/death, differentiation, migration, and adhesion. Previous studies have shown that the administration of C14 and P8 compounds [1] cause a strong binding of the mutant K-Ras4B/PDE6δ complex [2] inhibiting either it dissociation [3], or, and binding to the membrane [4] resulting in a diminution in the activation of its principal effectors Akt and Erk (red arrows). Modified from Refs. [8, 9].

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 KRAS gene. These mutations hinder GTP hydrolysis, resulting in continuous activation of the K-Ras protein and abnormal activation of K-Ras signaling pathways. Consequently, this abnormal activation promotes cell proliferation, differentiation, and survival (Figure 1) [10].

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 in silico (Figure 2) [17]. These molecules showed excellent results against KRAS-dependent cancers, with elevated affinity towards mutated variants of K-Ras, thereby preserving the signaling activity of K-Ras WT [8, 18, 19, 20]. After several assays carried out in a big library of compounds, the molecules denominated as C14 and P8 showed the best ability to specifically bind to and stabilize the mutated K-Ras4B/PDE6δ complex (Figure 2) [17].

Figure 2.
Anti-cancer compounds identified in-silico search. (A) 2-[(3-chlorophenyl) methyl-methyl-amino]-N-chroman-4-yl-acetamide structure known as C14. (B) 2-[4-(3-chlorophenyl)piperazin-1-yl]-N-[(4R)-chroman-4-yl]acetamide structure known as P8. (C and D) By molecular dynamic assays, both molecules demonstrated strong binding and stabilization of the most common mutant K-Ras4B G12D/PDE6δ complex. The dissociation of this complex is impaired and the GTPase function of K-Ras4B inhibited. Both compounds bind to the complex in different positions, which could have a synergic effect [8].

The antitumoral efficacy of these molecules has been assessed in various in silico models, in vitro models (cell lines and primary cultures), and in vivo models of pancreatic and colon cancer [8, 18]. These have resulted in the decreased activity of the principal K-Ras signaling pathways (e.g., MAPK and PI3K/Akt pathways) (Figures 1 and 2), apoptosis-mediated cancer cell death, reduction in clonogenic capacity and decreased tumor volume [8, 17].

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.

In vitro models play a vital role in drug discovery for cancer, as they afford a high degree of control over numerous variables within a research study, and the experimental design is less complex. Moreover, these models offer a cost-effective approach and serve as alternatives to animal use, thereby addressing ethical considerations [21]. In cancer drug discovery, in vitro models include various cell culture techniques, with three primary types demonstrating progressive degrees of complexity: 2D, primary, and 3D cultures. These advancements have significantly contributed to our understanding of cancer cells growth and migration, disease response, and drug interactions within the cell [22]. Currently, drug discovery and testing involve the use of both in vitro models and in vivo-animal models.

This chapter reviews the most common in vitro models used to characterize and validate the efficacy of C14 and P8 as the main drugs against the K-Ras/PDE6δ complex. An overview of cell cultures is provided, including cell lines in 2D conditions, 3D systems, and primary cell cultures.

2. In vitro models for anti-cancer drug discovery

The process of drug development and its evaluation can indeed be costly. As a result, in vitro cell culture models have been extensively utilized for these purposes due to their numerous benefits, including reduced expenses and time compared to animal studies. These in vitro models can be classified into two types: 2D and 3D models, each with its own set of advantages and disadvantages, as described in Table 1. Furthermore, the source from which the cells used as models are obtained can be adapted to the study’s characteristics. Therefore, the combination of cell lineage or culture conditions provides great versatility to in vitro models. For instance, when screening and selecting leads from hundreds of compounds, a 2D assay would be the best option over 3D models, as it allows for the lowest feasible cost due to the reduced requirement of cells and time for these assays. However, for fewer compounds requiring more in-depth characterization, a 3D model would be more suitable [23].

	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 in vivo conditions	Closer to in vivo physiological conditions	Closer to in vivo physiological conditions and structures 3D cell culture models more closely mimic in vivo behavior and better represents drug metabolism. Additionally, protein and gene expression levels in culture resembling those found in vivo.
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.

Table 1.

Advantages and disadvantages of in vitro culture methods.

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 in vitro systems typically established and propagated in a growth medium in tubes, flasks, or dishes as a monolayer culture. Here, cells can continue to divide indefinitely. Using a cell line allows each cell in an experiment to have a similar response because genetic variation is minimal. This can help researchers to observe differences between test groups more clearly [25].

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.

Table 2.

Cell lines employed for drug screening.

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 KRAS (>90%) [28]. Among the muted forms that are present in PDAC, the most frequently are G12D (~51%), G12V (~32%) and G12R (~12%) [29]. These mutations have been associated with a poor prognosis due to their ability to induce alterations in cancer cells, enabling them to evade immune response, reprograming their metabolism, and developing resistance to therapy [30]. Given the importance of KRAS mutations in the carcinogenesis and tumor progression of PDAC, mutant pancreatic cell lines are an excellent in vitro model for anti K-Ras/PDE6δ drug testing. In our research group, we evaluated the antitumoral effects of the candidates molecules in the pancreatic cell lines (ATCC, USA) as describe in Figure 3.

Figure 3.
Representative images of principal pancreatic cancer cell lines that present most of the KRAS mutations. In brief, BxPc-3 is a pancreatic cell line, which does not express the mutant form of K-Ras4B (Figure 1B). Panc-1 expresses the mutation in exon 2 of the KRAS gene, K-Ras4B^G12D (Figure 1C). MIA-PaCa expresses the mutation in exon 2 of the KRAS gene, K-Ras4B^G12C (Figure 1D) and CAPAN-1 expresses the mutation in exon 2 of the KRAS gene, K-Ras4B^G12V. The non-tumoral pancreatic cell line hTERT HPNE, was employed as control (Figure 1A). Scale bar 200 μM.

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 KRAS play an important role in carcinogenesis or progression of BC [33, 34] and, this gene is mutated in about 7%-12% of the patients (2% luminal A, 20% luminal B, 17% HER2, 0–8% TNBC) [3]. Specifically, the presence of oncogenic K-Ras is associated with the more aggressive luminal B and HER2 subtypes compared to the luminal A type [35]. Significantly, this gene is amplified in approximately 32% of TNBC cases [36] leading to an upregulation of KRAS signaling pathways and an enhanced tumor microenvironment that promotes tumor progression [33]. Breast cancer cell lines are described in Figure 4.

Figure 4.
Representative images of breast cancer cell lines. Breast cancer cell lines and derived radio-resistant cells growth in monolayer. Scale bar 200 μM. In brief, MCF-7 is an estrogen receptor (ER)-positive cell line, which does not express the mutant form of K-Ras4B (Figure 3B). MDA-MB-231 is a TNBC that expresses the mutation in exon 2 of the KRAS gene, K-Ras4B^G13D (Figure 5C) [37]. To explore the effect of C14 and P8 over radioresistant cell lines, MCF 7RR which does not contain the mutation in K-Ras4B (Figure 3D) and MDA-MB-231RR, that possess the mutation K-Ras4BG13D as was observed in its parental cell line were used (Figure 5D) and MDA-MB-231RR, that possess the mutation K-Ras4BG13D as was observed in its parental cell line (Figure 5E) [38]. The non-tumoral breast cell line MCF 10A, was employed as control (Figure 5A).

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 in vitro models because it indicates the amount of a drug is required to inhibit a biological process by half, indicating changes in the population due to increased cell death or decreased cell proliferation. In cancer, using the IC50 concentration of a compounds means killing cancer cells and preventing cancer cell growth while having a less damage on healthy cells in the body or inhibiting tumor growth by half. A low IC50 suggests that the drugs will be effective at lower concentrations and will consequently cause less systemic toxicity when administered to patients for therapy [42].

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).

Figure 5.
Dose-response curves of pancreatic cell lines treated with compounds C14 and P8. Dose-response curves using 0–200 μM concentrations of compound C14 and P8 tested at 48 hours post incubation measured with MTT. Each curve was done by three independent replicates.

Another crucial point is the specific effect of C14 and P8 over malignant cells. As the results showed above, KRAS mutant cell lines Panc-1-G12D, MIA-PaCa-G12C and Capan-1-G12V exhibit a considerable cytotoxic effect for both compounds compared to the vehicle (DMSO) (Figure 5C, D and E). This behavior was also observed in BxPC-3WT cell line, although BXPC-3 cells are WT for KRAS, the present several other mutations, which contribute to the activation of K-Ras and the dependence to other effectors downstream K-Ras signaling pathway, potentially explaining their susceptibility to the treatment [43].

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 KRAS mutant cancer cells and highly KRAS-dependent cells without affecting healthy cells, making them a promising treatment alternative.

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.

Figure 6.
Dose-response curves of the breast cell lines treated with compounds C14 and P8. Dose-response curves using 0–200 μM concentrations of compound C14 and P8 tested at 72 hours post incubation measured with XTT each curve was done by three independent replicates.

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 KRAS-mutant cells such as, MDA-MB-231 (Figure 6C). This discovery was also observed in the radioresistant cell line MDA-MB-231RR, where the compounds also had a significant inhibitory effect at 72 hours (Figure 6D). This finding suggests an alternative course of treatment for patients who have experienced a cancer recurrence after radiation therapy. Finally, in the case of MCF 7 and MCF 7RR, the IC50 values in them were higher than those found in KRAS mutant lines (Figure 6B and E). It is important to note that the compounds have an inhibitory effect on the viability of luminal and radioresistant breast cancer cells, even though that these cell lines do not carry the KRAS gene mutation (Figure 6A, E and F).

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.

Table 3.

Different methods to obtain primary cultures from pancreatic cancer.

Figure 7.
Representative image of the primary cell cultures after three passages denominated as MGKRAS004 (A) and MGKRAS005 (B), obtained from malignant pancreatic tumors. Scale bar (red line) 100 μM.

Figure 8.
Expression of CSC markers in primary pancreatic cultures. (A) CD44 expression in MGKRAS004 and MGKRAS005 cells. (B) CD24 expression in MGKRAS004 and MGKRAS005 cells. (C) CD133 expression in MGKRAS004 and MGKRAS005 cells.

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).

Figure 9.
Cancer stem cell proportion in pancreatic primary culture. (A) Quantification by FACS of CD44+, CD24+ and CD133+ in 2D conditions of pancreatic cancer primary cultures. (B) Quantification by FACS of CD44+, CD24+ and CD133+ in 3D conditions of pancreatic cancer primary cultures. (C) Comparison of % of CSC in 2D and 3D conditions.

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	—

Table 4.

Nutritional requirements for primary pancreatic cultures since its insolation and placed culture passage 0 (P0) until passages 4 to 10 (P4–10).

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].

Figure 10.
Effect on the viability of primary cultures in 2D. (A) MGKRAS004 in 2D treated with C14 and P8. (B) MGKRAS005 in 2D treated with C14 and P8.

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. In vitro models, plays an important role in drug discovery, especially in the early and preclinical stages. Considering this, despite the increase in the IC50 value in pancreatic primary cultures these compounds shown a significant cytotoxic effect in cancer cells, making them candidates for more in-depth characterization.

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) in vitro models have been developed. In this models, it has been possible to reproduce a more detailed aspects of tumoral behavior including interactions among tumor cells, the extracellular matrix, and stromal cells [57]. Additionally, cells growing in 3D, conditions have been exhibited a greater resistance to chemotherapeutics agents and mayor invasiveness compared to 2D models [58, 59]. As a result, the use of 3D models has been gaining more popularity every day. Considering this, we present the advances obtained in 3D models to evaluate the effect of C14 and P8 in one more realistic pancreatic cancer cells systems.

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).

Figure 11.
Representative images of spheroids from cell lines (MDA-MB-231, MDA-MB-231 RR, MCF7 and MCF 7RR) and primary cell cultures (MGKRAS004 and MGKRAS005) after 7 days of culture. Scale bar 200 μM.

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 in vivo microenvironment of the patient’s tumor [69].

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).

MFE(%)=(number of mammospheres per well) (number of cells seeded per well) ×100E1

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.

Figure 12.
Effect on the viability of primary cultures in 3D, treated with C14 and P8. (A) MGKRAS004 in 3D treated with C14 and P8. (B) MGKRAS005 in 3D treated with C14 and P8.

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 in vitro and in vivo models prior to patient administration [24]. This chapter presents the assessment of compounds C14 and P8 in diverse models. The data presented supports the efficacy of both molecules in mitigating the adverse effects induced by various mutant forms of K-Ras in both pancreatic and breast models. However, we encountered several challenges in stablishing the effectiveness of these molecules. One significant challenge was selecting the appropriate method to determine the IC (inhibitory concentration) of each compound. Conventional methods using XTT and/or MTT as cell proliferation quantification techniques have limitations since their response relies on the metabolic rate of the specific cell lines used, which may not accurately reflect the true growth rate of the models being studied. To address this issue, we recognize the importance of implementing models that measure cell growth by quantifying elements independent of cellular metabolism, such as DNA/RNA or ATP [77].

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

In vitro models have been of great importance in cancer research and drug screening. The use of these models allows the study of numerous cell process, the effect of drugs on cells, and the processes that trigger apoptosis.

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 in vivo, resulting in many drug candidates failing during clinical trials.

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 vivo conditions, with protein and gene expression similar to those found in vivo. Due to these advantages, 3D primary cell culture has gained popularity and holds the potential to replace animal in vivo models in future, eventually leading to direct human clinical trials.

In conclusion, this chapter reviews several in vitro models for assessing of anti-cancer compounds, demonstrating their selective effectiveness in reducing viability of pancreatic and breast cancer cells in different conditions. These findings are of vital importance, as current chemotherapies often result in severe side effects. The pursuit of alternative therapies with fewer adverse effects could greatly benefit patients with these pathologies. Considering the evidence presented, we propose that compounds C14 and P8 represent novel therapeutic alternatives for the treatment of tumors dependent on oncogenic forms of K-Ras.

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.

Conflict of interest

The authors declare no conflict of interest.

Sunil Bhat

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