Research ArticleExperimental Studies

β-Tubulin Isoforms Related to Docetaxel Sensitivity in 2D and 3D Cultured TNBC Cell Lines

MASAKO MUGURUMA, MARIKO ASAOKA, SAEKO TERAOKA, KANA MIYAHARA, TAKAHIKO KAWATE and TAKASHI ISHIKAWA
Anticancer Research October 2022, 42 (10) 4735-4742; DOI: https://doi.org/10.21873/anticanres.15978

Abstract

Background/Aim: We previously reported that the half maximal inhibitory concentration (IC50) values of cisplatin and epirubicin correlated in 13 triple-negative breast cancer (TNBC) cell lines between two-dimensional (2D) and three-dimensional (3D) culture methods. However, the IC50 values of docetaxel (DTX) did not correlate between the two culture methods. We hypothesized that this non-correlation is partly associated with differences in expression of the β-tubulin isoform, the target molecule of DTX and in morphology depending on the culture method. Materials and Methods: We investigated the expression levels of β-tubulin isoforms by real-time polymerase chain reaction and morphology of spheroid formation in the 13 TNBC cell lines cultured using the 2D and 3D culture methods. Results: Tubulin β class I (TUBB) expression levels were negatively correlated with the IC50 value of DTX in the 2D culture method (R=−0.360), whereas tubulin β class IIa (TUBB2a) expression levels were positively correlated in the 3D culture method (R=0.398). There was no significant difference in the expression levels of β-tubulin isoforms between the 2D and 3D culture methods. The spheroids were classified morphologically into three types: round, mass, and grape-like. However, no clear association was found between DTX sensitivity and morphology. Conclusion: The non-correlation of the IC50 values of DTX between the 2D and 3D culture methods does not appear to be due to the changes in β-tubulin isoforms. Morphology in the 3D culture method may play some role in drug sensitivity.

Key Words:

We reported a correlation between the half maximal inhibitory concentration (IC50) values from the 2D and 3D cultures and the drug sensitivities of cisplatin (CDDP), epirubicin (EPI), and docetaxel (DTX). However, in the sensitivity assay for DTX, there was no correlation between the IC50 values of the two culture methods (1). The results of our clinical studies to date have suggested that the expression of class III β-tubulin is a contributing factor to the resistance of triple-negative breast cancer (TNBC) to DTX (2). Therefore, we hypothesized that the correlation between the two culture methods and the DTX IC50 involved the expression of class III β-tubulin. Docetaxel, like paclitaxel, binds to β-tubulin and increases microtubule formation by increasing microtubule polymerization and inhibiting depolymerization, thereby causing abnormal microtubule dynamics (3).

There are 9 β-tubulin isoforms in humans, namely, tubulin β class I (TUBB), tubulin β1 class VI (TUBB1), tubulin β2A class IIa (TUBB2A), tubulin β2B class IIb (TUBB2B), tubulin β3 class III (TUBB3), tubulin β4A class IVa (TUBB4A), tubulin β4B class IVb (TUBB4B), tubulin β6 class V (TUBB6), and tubulin β8 class VIII (TUBB8) (4). TUBB is the most predominantly expressed isotype in human tumor cell lines, including breast cancer (5). In female patients with colorectal cancer, the expression of TUBB3, TUBB6, and androgen receptor is reportedly associated with poor prognosis (6). High expression of TUBB1, TUBB2, and TUBB3 has been reported to be associated with unfavorable clinicopathologic factors in urothelial carcinoma of the bladder (7).

Cells grow only when they attached to the bottom of the plate in 2D culture, and necrotic cells are removed on medium exchange. Thus, a 2D culture environment is completely different from the normal physiological conditions and the results of in vitro drug sensitivity tests are not always consistent with clinical outcomes (8). In vitro experimental data suggest that spheroids represent physiological tumors better than cell monolayers (9). A 3D spheroid model can re-establish the morphological, functional, and mass transport properties of the corresponding tissue in vivo. There are two main methods of 3D culture, namely, scaffold and scaffold-free systems. The scaffold-free system is the most widely used model because it does not require a support for cell growth, and it is suitable for the culture of tumor cells (10). Spheroid morphology and drug sensitivity have been reported in breast (11), and prostate (12). Recently, studies based on spheroid size have reported a close correlation with biochemical analysis of drug response in spheroid (13).

In the present study, we investigated the gene expression of the 5 β-tubulin isoforms to clarify the differences in the DTX sensitivity of TNBCs cultured using 2D and 3D methods. We also investigated the spheroid morphology and proliferative potential of the 13 TNBC cell lines under 3D culture in relation to drug resistance.

Materials and Methods

Cell culture and reagents. Twelve cell lines were obtained from the American Type Culture Collection (ATCC: Manassas, VA, USA). These include HCC1599, HCC1937, HCC1395, HCC70, HCC1806, HCC38, BT549, Hs578T, BT20, MDA-MB-468, MDA-MB-231, and MDA-MB-436. One cell line (i.e., MDA-MB-453) was obtained from the RIKEN BioResource Center Cell Bank (RIKEN BRC Cell Bank; Tsukuba, Ibaraki, Japan). HCC1599, HCC1937, HCC1395, HCC70, HCC1806, HCC38, BT549, Hs578T, and BT20 were maintained in RPMI 1640 medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaillé, France), with 10,000 units penicillin, 10 mg streptomycin, and 25 μg amphotericin B/mL (Sigma-Aldrich®, St. Louis, MO, USA), 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES, pH 7.0–7.6, Sigma-Aldrich®), and 1 mM sodium pyruvate solution (Sigma-Aldrich®) at a constant temperature of 37°C in a humidified atmosphere of 5% CO2 and 95% air. The MDA-MB-468, MDA-MB-231, MDA-MB-453, and MDA-MB-436 were maintained in Leibovitz’s L-15 medium (Life Technologies Corporation®, Grand Island, NY, USA) supplemented with 10% FBS (Biowest) and 10,000 units penicillin, 10 mg streptomycin, and 25 μg amphotericin B/ml (Sigma-Aldrich®) in a culture flask at a constant temperature of 37°C in 100% air.

Specimen preparation for total RNA extraction from 2D- and 3D-cultured TNBC cell lines. To prepare 2D-cultures, the 13 TNBC cell lines were seeded at the optimal number of cells (Table I) in a 10 cm tissue culture dish (Corning Incorporated, Corning, NY, USA) and cultured for 5 days. To isolate total RNA from the 2D-cultured cells, the culture medium was aspirated from subconfluent cells, and Lysis Buffer RA1 (MACHEREY-NAGEL, Düren, Germany) with β-Mercaptoethanol (FUJIFILM Wako Pure Chemical Corporation) was immediately added to the cell culture dish and the cells were completely lysed.

View this table:
Table I.

Number of seeded cells for the 2D and 3D culture plate conditions of the 13 triple-negative breast cancer (TNBC) cell lines used in this study.

For the 3D culture, all TNBC cell lines were seeded on an ultra-low attachment 96-well cell culture plate (PrimeSurface® 96U or 96V; Sumitomo Bakelite, Tokyo, Japan) at 2,000 cells/100 μl/well and cultured for 5 days. Details of the culture conditions used in the 3D culture are listed in Table I. On culture day 5, all the spheroid cells from the ultra-low attachment cell culture plate and culture medium were collected in a 50 ml centrifuge tube (Corning Incorporated) and centrifuged at 250 × g for 5 min. The supernatant was carefully aspirated and RA1 was added to the spheroid cells. The 2D- and 3D-cultured cell specimens in RA1 were stored at −80°C until total RNA extraction.

Drug sensitivity assay. Drug sensitivity testing of DTX using 2D and 3D cultured TNBCs was performed as previously reported (1). Briefly, for 2D culture, 12 adhering TNBC cell lines were seeded on a 96-well flat-bottomed cell culture plate at 2,000 cells/100 μl/well/96 well culture for 24 hours. Following overnight incubation, cells were sensitized in optimal culture media with serially diluted DTX for 120 h. As for the suspension cell line HCC1599, cells were plated at 2,000 cells/75 μl/well/96 well culture and sensitized with DTX at the initial seeding. The cell viability in 2D culture was determined by measuring cellular ATP level using the CellTiter-Glo 2.0. (Promega, Madison, WI, USA). The procedure was performed according to the manufacturer’s instructions. Then, relative luminescence units were measured using the EnSpire Multi-mode plate reader (PerkinElmer, Waltham, MA, USA).

For 3D culture, all TNBC cell lines were seeded on an ultra-low attachment cell culture plate (Sumitomo Bakelite) at 2,000 cells/100 μl/well/96 well culture for 72 h. After forming spheroids at 72 hours, DTX exposure was performed similarly to the 2D culture. Cellular ATP level was measured using the CellTiter-Glo 3D (Promega Corp.).

RNA purification from cultured cells, cDNA synthesis and quantitative real-time PCR. Frozen specimens of the 2D- and 3D-cultured cells lysed with RA1 were slowly thawed before starting total RNA isolation. Total RNA was extracted using the NucleoSpin® RNA kit according to the manufacturer’s protocol (MACHEREY-NAGEL). cDNA was synthesized from 300 ng total RNA/specimen using the SuperScript™ II 1st strand cDNA Synthesis Kit (Takara Bio., Shiga, Japan) with random primers in a 20 μl reaction mixture. Quantitative real-time RT-PCR with FastStart Essential DNA Green Master (Roche Diagnostics, Mannheim, Germany) was performed using the LightCycler® 96 (Roche Diagnostics). The real-time RT-PCR reaction was performed according to the FastStart Essential DNA Green Master mix protocol. RNA expression levels were analyzed using the ΔΔCt method and normalized to the values of β-actin. The same experiment was repeated at least two times. The sequences of the primers are listed in Table II.

View this table:
Table II.

Sequences of primers using real-time RT-PCR analysis.

Spheroid morphological analysis. Images of spheroid cells were observed and recorded using an inverted phase-contrast microscope (AXIO Vert.A1; Carl Zeiss Microscopy, Jena, Germany). The spheroid area of each cell line was measured and recorded on the first and fifth days of culture using the ZEN pro analysis software (Carl Zeiss).

Statistical analysis. Statistical analyses were carried out using Microsoft® Excel® 2016 MSO (Version 2206 Microsoft, Redmond, WA, USA). Results are expressed as mean±standard deviation of n independent experiments. All experiments were performed at least twice and then statistically compared using Student’s t-test. A p-value of less than 0.05 was considered to indicate a statistically significant difference between 2 groups.

Results

Gene expression analysis of β-tubulin isoforms. In the 2D-cultured TNBC cell lines, there was a weak negative correlation between the IC50 values of DTX and the expression levels of TUBB (R=−0.360, Figure 1A), but there was no correlation between the IC50 values of DTX and the expression levels of TUBB2a (Figure 1C), TUBB3 (Figure 1E), TUBB4b (Figure 1G), and TUBB6 (Figure 1I). In the 3D-cultured TNBC cell lines, there was a weak positive correlation between the IC50 values of DTX and the expression levels of TUBB2a (R=0.398, Figure 1D), but there was no correlation between the IC50 value of DTX and the expression levels of TUBB (Figure 1B), TUBB3 (Figure 1F), TUBB4b (Figure 1H), and TUBB6 (Figure 1J).

Figure 1.
Figure 1.

Correlation analysis between IC50 values of DTX and β-tubulin isoform expression in 2D- and 3-cultured triple-negative breast cancers. Columns on the left and right show the 2D- and 3D-TNBCs, respectively. The x axis shows the IC50 value of DTX. The y axis shows the expression levels of TUBB isoforms obtained from qPCR based on β-actin.

As the IC50 values of DTX in the 2D and 3D cultures of HCC1806, HS578T, HCC70, and MDA-MB-231 cell lines differed by more than 10-fold (1), we compared the differences in the β-tubulin isoform expression in these cell lines between the 2 culture methods. In HCC1806 cells, the expression levels of TUBB (Figure 2A, p=7.5×10−5), TUBB2a (Figure 2B, p=1.2×10−4), TUBB3 (Figure 2C, p=0.021), TUBB4b (Figure 2D, p=9.6×10−6), and TUBB6 (Figure 2E, p=0.002) were significantly up-regulated in the 3D culture compared with the 2D culture. In HCC70 cells, the expression levels of TUBB (Figure 2A, p=1.7×10−7), TUBB3 (Figure 2C, p=0.014), and TUBB6 (Figure 2E, p=0.006) were significantly up-regulated in the 3D culture compared with the 2D culture. In Hs578T cells, the expression levels of TUBB2a (Figure 2B, p=2.9×10−4) and TUBB6 (Figure 2E, p=0.002) were significantly down-regulated in the 3D culture compared with the 2D culture. In MDA-MB-231 cells, the expression levels of TUBB (Figure 2A, p=1.3×10−10), TUBB2a (Figure 2B, p=1.7×10−14), TUBB4b (Figure 2D, p=1.2×10−20) and TUBB6 (Figure 2E, p=8.2×10−16) were significantly down-regulated, and the expression levels of TUBB3 (Figure 2C, p=2.0×10−4) were significantly up-regulated in the 3D culture compared with the 2D culture.

Figure 2.
Figure 2.

Comparison of β-tubulin isoform expression levels between 2D and 3D cultures in each of 13 triple-negative breast cancer cell lines. (A) TUBB, (B) TUBB2a, (C) TUBB3, (D) TUBB4b, and (E) TUBB6. Data represent the mean±standard deviation of the two independent experiments. Number of samples (n) as noted in the graph. p-values (p) were calculated by Student’s t-test.

The 3D cultured HCC1937 and HCC1395 cells showed strong resistance to DTX (1). In HCC1937 cells, the expression levels of TUBB (Figure 2A, p=2.2×10−4), TUBB4b (Figure 2D, p=0.002), and TUBB6 (Figure 2E, p=0.006) were significantly down-regulated in the 3D culture compared with the 2D culture. In HCC1395 cells, the expression levels of TUBB2a (Figure 2B, p=8.7×10−5) and TUBB3 (Figure 2C, p=0.005) were significantly down-regulated in the 3D culture compared to the 2D culture.

The results of the comparison of the β-tubulin isoforms TUBB, TUBB2a, TUBB3, TUBB4b, and TUBB6 are shown in Figure 3A-E. The expression levels of TUBB (Figure 3A, p=0.724) and TUBB3 (Figure 3C, p=0.789) did not change markedly between the two culture methods. The expression levels of TUBB2a (Figure 3B, p=0.332) showed an increase in the 3D culture compared with the 2D culture, but the change was not significant. The expression levels of TUBB4b (Figure 3D, p=0.468) and TUBB6 (Figure 3E, p=0.463) showed a decreasing trend in the 3D culture compared with the 2D culture, but the change was not significant.

Figure 3.
Figure 3.

Comparison of β-tubulin isoform expression levels between 2D and 3D cultures in 13 triple-negative breast cancer cell lines. (A) TUBB, (B) TUBB2a, (C) TUBB3, (D) TUBB4b, and (E) TUBB6. Data represent the mean±standard deviation of two independent experiments. p-values (p) were calculated by Student’s t-test.

Morphology and cell growth of 3D-cultured TNBC cell lines. The morphology of the spheroids was classified into 3 types: round, mass, and grape-like. HCC38, HCC1937, HCC70, Hs578T, HCC1395, and BT20 cell spheroids were classified as round type. Their typical images are shown in Figure 4A. The spheroid areas of HCC1937 (6.388-fold, p=9.4×10−7), HCC70 (1.925-fold, p=0.0017), HCC1395 (13.720-fold, p=5.1×10−4), and BT20 (1.839-fold, p=2.0×10−7) cells showed a significant increase after 5 days of culture. There was no significant increase in the spheroid area of HCC38 cells (1.047-fold, p=0.639) during 5 days of incubation. The spheroid area of Hs578T cells (0.855-fold, p=0.0028) showed a significant decrease after 5 days of culture.

Figure 4.
Figure 4.

Typical morphology images and cell growth of 3D-cultured 13 triple-negative breast cancer cell lines. (A) Round type by spheroid morphology classification. (B) Mass type by spheroid morphology classification. (C) Grape-like type by spheroid morphology classification. The images show spheroid of each cell line on day 1 and day 5 after the start of 3D culture, and the spheroid cell area grown during the 5 days was measured and recorded using an inverted phase-contrast microscope and analyzed using the ZEN pro analysis software. Number of samples (n), spheroid area (mm2), p-value (p) and SD (standard deviation) are described in each cell lines.

The spheroids of MDA-MB-468, HCC1806, BT549, MDA-MB-231, MDA-MB-436, and MDA-MB-453 cells were classified as mass type. Their typical images are shown in Figure 4B. After 5 days of incubation, MDA-MB-468 (2.620-fold, p=2.0×10−13), HCC1806 (1.412-fold, p=4.4×10−4), MDA-MB-231 (2.194-fold, p=4.5×10−9), MDA-MB-436 (1.324-fold, p=5.2×10−5), and MDA-MB-453 (2.451-fold, p=3.3×10−9) cells showed a significant increase, whereas BT549 cells (0.996-fold, p=0.995) showed no significant increase in the spheroid area.

The spheroids of HCC1599 cells were classified as Grape-like type and is shown in Figure 4C. The spheroid area of HCC1599 cells showed a significant increase (1.450-fold, p=0.038).

Discussion

We found that the IC50 values of DTX were negatively correlated with TUBB expression in 2D-cultured TNBCs and positively correlated with TUBB2a expression in 3D-cultured TNBCs (R=−0.360 and R=0.398, respectively). It is possible that there is some relationship between β-tubulin isoform expression and DTX sensitivities. However, no significant differences were found in the expression of β-tubulin isoforms between these two culture methods. Therefore, the different sensitivities to DTX between the two culture methods cannot be fully explained by β-tubulin isoform expression patterns.

Previously, the associations of both protein and mRNA of β-tubulin isoforms with DTX sensitivity were investigated in two breast cancer cell lines. The mRNA expression patterns of β-tubulin isoforms were not always consistent with protein expression (5). A significant correlation was previously found between protein expression of class III β-tubulin and sensitivity to tubulin-binding agents in kidney cancer cell lines (14) and a prostate cancer cell line (15). Thus, observance of β-tubulin protein expressions rather than mRNA may be more advantageous in investigating the association of β-tubulin with DTX sensitivity.

We observed three morphological types of spheroids consistent with previous reports: round, mass, and grape-like (1, 16). Spheroid morphologies are reportedly associated with drug sensitivity to DTX. Round-shaped spheroids showed no change in shape after DTX treatment, whereas mass-shaped spheroids lost their morphology (17). In the present study, HCC1395 and HCC1937 (highly resistant to DTX) (18, 19) cells formed large and round spheroids. Large tumor spheroids have been reported to be more heterogeneous than small tumor spheroids Proliferating cells are located in the outer regions and quiescent cells in the inner regions because the conditions for nutrient and gas exchange are different in each layer (20). Although a clear association between morphology and DTX sensitivity was not found, the morphology and size of spheroids are thought to be involved in drug resistance in the 3D culture method.

In conclusion, differences in sensitivity to DTX between the 2D and 3D culture methods could not be fully explained by β-tubulin isoform gene expression. The association of the morphological characteristics of spheroids with drug sensitivity therefore warrants further investigation.

Acknowledgements

The Authors thank Dr. Edward Barroga (http://orcid.org/0000-0002-8920-2607) for reviewing and editing the article. The Authors also thank the Medical Research Center of Tokyo Medical University for the use of research equipment.

Footnotes

  • Authors’ Contributions

    M. Muguruma performed the majority of the experiments. M. Muguruma analyzed and interpreted data and wrote the initial manuscript together with M. Asaoka, and T. Ishikawa. M. Asaoka, S. Teraoka, K, Miyahara and T, Kawate reviewed and revised the manuscript for important contents. T. Ishikawa was responsible for the overall study concept, design, write-up, data analysis, data interpretation, and funding acquisition. All authors contributed to writing and revising the manuscript.

  • Conflicts of Interest

    The Authors declare that they have no competing interests in relation to this study.

  • Received July 19, 2022.
  • Revision received August 3, 2022.
  • Accepted August 6, 2022.

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