Analysis of Multiple Drug Resistance Mechanism in Different Types of Soft Tissue Sarcomas: Assessment of the Expression of ABC-Transporters, MVP, YB-1, and Analysis of Their Correlation with Chemosensitivity of Cancer Cells

Abstract

Chemotherapy of soft tissue sarcomas (STS) is restricted by low chemosensitivity and multiple drug resistance (MDR). The purpose of our study was the analysis of MDR mechanism in different types of STS. We assessed the expression of ABC-transporters, MVP, YB-1, and analyzed their correlation with chemosensitivity of cancer cells. STS specimens were obtained from 70 patients without metastatic disease (2018-2020). Expression level of MDR-associated genes was estimated by qRT-PCR and cytofluorimetry. Mutations in ABC-transporter genes were captured by exome sequencing. Chemosensitivity (SI) of STS to doxorubicin (Dox), ifosfamide (Ifo), gemcitabine (Gem), and docetaxel (Doc) was analyzed in vitro. We found strong correlation in ABCB1, ABCC1, and ABCG2 expression. We demonstrated strong negative correlations in ABCB1 and ABCG2 expression with SI (Doc) and SI (Doc + Gem), and positive correlation of MVP expression with SI (Doc) and SI (Doc + Gem) in undifferentiated pleomorphic sarcoma. Pgp expression was shown in 5 out of 44 STS samples with prevalence of synovial sarcoma relapses and it is strongly correlated with SI (Gem). Mutations in MDR-associated genes were rarely found. Overall, STS demonstrated high heterogeneity in chemosensitivity that makes reasonable in vitro chemosensitivity testing to improve personalized STS therapy, and classic ABC-transporters are not obviously involved in MDR appearance.

Keywords: ABCB1; ABCC1; ABCG2; MVP; Pgp; YB-1; chemosensitivity test; soft tissue sarcoma; synovial sarcoma; undifferentiated pleomorphic sarcoma.

Figure 1

Examples of cytological studies of the derived cell cultures (Leishman stain; 80×). (A) Cell culture obtained from the patient with synovial sarcoma (99% malignant cells), (B) cell culture obtained from the patient with malignant schwannoma (20% malignant cells, 80% lymphocytes), (C) cell culture obtained from the patient with undifferentiated pleomorphic sarcoma (99% malignant cells), (D) cell culture obtained from the patient with liposarcoma (99% of malignant cells).

Figure 2

Frequency histograms of the sensitivity index for each single drug (Dox, Ifo, Doc, Gem) and two combinations (Dox + Ifo, Doc + Gem).

Figure 3

P-gp expression analysis by cytofluorometry. (A) K562/i-S9 cells were stained with FITC-labeled antibodies against Pgp (Pgp expressed by 98.2% cells); K562/i-S9 cell subline were obtained from chronic myeloid leukemia cell line K562 by transduction of MDR1 (ABCB1). K562/i-S9 cells were used as positive control for the determination of Pgp staining. (B) Expression of Pgp in 33.8% of cells in STS 154 (synovial sarcoma). (C) Expression of Pgp in 8% of cells in STS 159 (leiomyosarcoma). (D) Expression of Pgp in 0.7% of cells in STS 161 (synovial sarcoma).

Figure 4

Validation of Pgp protein expression in STS samples by Western blotting. Lines 1–8: analyzed STS samples. Line 9: positive control of anti-Pgp antibodies activity (K562/i-S9 cells).

Figure 5

ABCG2 expression analysis by cytofluorometry. (A) HBL-100/Dox cells were stained with APC-labelled anti-ABCG2 antibodies (ABCG2 expressed by 50% cells). Dox-resistant cell subline HBL-100/Dox with overexpressed ABCG2 (BCRP) was obtained from the HBL-100 cells after the selection with Dox. HBL-100/Dox cells were used as positive control for the determination of ABCG2 staining. (B) Expression of ABCG2 in 17.1% of cells in STS 87 (liposarcoma). (C) Expression of ABCG2 in 8.6% of cells in STS 97 (undifferentiated pleomorphic sarcoma). (D) Expression of ABCG2 in 0.4% of cells in STS 90 (synovial sarcoma).