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

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.


Introduction
Success in chemotherapy of soft tissue sarcomas (STS) is still difficult to achieve due to multiple obstacles. STS are heterogeneous cancers with more than 100 histological subtypes, different in molecular abnormality profiles [1] that make investigations in the field of STS personalized therapy very complex [2]. In spite of the multiple ongoing clinical trials for

Evaluation of the Chemosensitivity of Primary Cultured STS Cells to Anticancer Drugs
We obtained 70 primary cell cultures from STS samples. At passages 0 and 2-3 we evaluated the percentage of tumor cells in the cultures with cytological staining. In further analysis we used the cultures with ≥90% of tumor cells (Figure 1).
We evaluated the resistance of cancer cells from STS primary cultures to doxorubicin (Dox), ifosfamide (Ifo), docetaxel (Doc), gemcitabine (Gem), and the combinations: (Dox + Ifo) and (Doc + Gem). If the sensitivity index (SI) was less than 300, cells were accounted as sensitive to the drug or drug combination. Figure 2 demonstrates the histograms of SI frequencies for all drugs individually and for drug combinations. It is noteworthy that primary cells revealed the high heterogeneity; therefore, the SI frequencies did not match to normal distribution. Resistant STS are more frequently occurred in case of Doc and Gem: SI > 300 in 74.3% and 67.1% of samples after individual treatment and 47.1% after combination treatment. In case of Dox and Ifo these indexes were 51.4% and 46.7%, respectively, and 25.9% for combination. We evaluated the resistance of cancer cells from STS primary cultures to doxorubicin (Dox), ifosfamide (Ifo), docetaxel (Doc), gemcitabine (Gem), and the combinations: (Dox + Ifo) and (Doc + Gem). If the sensitivity index (SI) was less than 300, cells were accounted as sensitive to the drug or drug combination. Figure 2 demonstrates the histograms of SI frequencies for all drugs individually and for drug combinations. It is noteworthy that primary cells revealed the high heterogeneity; therefore, the SI frequencies did not match to normal distribution. Resistant STS are more frequently occurred in case of Doc and Gem: SI >300 in 74.3% and 67.1% of samples after individual treatment and 47.1% after combination treatment. In case of Dox and Ifo these indexes were 51.4% and 46.7%, respectively, and 25.9% for combination. SIs of studied drugs demonstrated moderate positive correlation among themselves (r interval was 0.44-0.72, p < 0.0001). Strong correlation was shown for SI (Doc) and SI SIs of studied drugs demonstrated moderate positive correlation among themselves (r interval was 0.44-0.72, p < 0.0001). Strong correlation was shown for SI (Doc) and SI (Gem) with SI (Doc + Gem), r = 0.85 и r = 0.84, p < 0.0001, respectively, as well as between SI (Ifo) and SI (Dox + Ifo), r = 0.83, p < 0.0001. STS cells demonstrated higher resistance to Doc and Gem, than to Dox and Ifo (p < 0.001). Drug combinations were more effective than individual treatments (p < 0.01), and combination Dox + Ifo showed higher cytotoxicity than Doc + Gem (p = 0.03) ( Table 1). Forty-one percent of primary cell cultures were sensitive to both drug combinations, and 22% of them showed resistant phenotype. Furthermore, we observed higher resistance to Dox in tumor cells from the patients of the age above 40 years old (p = 0.01). The SI of other drugs were not associated with age, sex, tumor size and previously applied chemotherapy. We compared the SI level in undifferentiated pleomorphic sarcoma, liposarcoma, and synovial sarcoma as these groups contained the largest number of samples. Undifferentiated pleomorphic sarcoma cells were more resistance to Dox then synovial sarcoma (p = 0.01 for pairwise comparison and p = 0.04 for group comparison). We did not observe any correlation between SI of other drugs and tumor histology type. However, we showed an interesting association between SI (Doc) and tumor regression grade: groups of patients, who received adjuvant or neoadjuvant chemotherapy and demonstrated tumor regression grade of 2-3 level, have Doc-resistant tumor cells more frequently: SI (Doc) 304 ± 122 in group with 0-1 tumor regression grade vs. SI (Doc) 487 ± 74 in group with 2-3 tumor regression grade (p = 0.0014). Moreover, comparing the patient groups with neoadjuvant therapy only, SI (Doc), and SI (Doc + Gem) were higher in primary cell cultures from the patients with better response to the therapy (p = 0.008 and p = 0.03, respectively) ( Table 2).

Expression of MDR-Associated Genes in STS
We analyzed the relative expression of ABC-transporters' genes ABCB1, ABCC1, and ABCG2, transcription factor YB-1 and MVP gene associated with the distribution of xenobiotics in cytoplasm, in 70 tumor samples. We revealed the strong positive correlation between ABCB1/ABCC1 and ABCB1/ABCG2 expression and between ABCC1/ABCG2 expression. MVP expression demonstrated weak positive correlation with YB-1 and weak negative correlation with ABCB1. YB-1 expression did not correlate with the expression of ABC-transporters (Table 3).  We observed higher ABCG2 expression level (p = 0.007) in patients at the age above 40 years old. Expression of other studied genes were not associated with age, sex, tumor size, degree of malignancies and tumor regression grade (Table 4). In case of undifferentiated pleomorphic sarcoma YB-1 expression was higher than in liposarcoma samples (p = 0.009 for pairwise comparison, p = 0.03 for group comparison). In relapsed patients we observed the decreased expression of MVP (p = 0.04).
Generally, expression of studied genes did not correlate with the drug resistance (SI), except for weak negative correlation for ABCB1 expression and SI (Doc), r = −0.26; p = 0.02. MVP expression demonstrated weak positive correlation with SI (Doc), r = 0.24; p = 0.04.
Then we analyzed the correlation between expression of MDR-associated genes and SIs of chemotherapeutics in three groups of STS of different histological subtypes: undifferentiated pleomorphic sarcoma (24 samples), liposarcomas (16 samples), synovial sarcomas (11 samples). In liposarcomas we did not show any correlations in SIs of studied drugs. In undifferentiated pleomorphic sarcoma we demonstrated the medium negative correlation of ABCB1 and ABCG2 expression with SI (Doc) and SI (Doc + Gem). In further analysis we showed that these correlations were more pronounced in the group of undifferentiated pleomorphic sarcoma relapses. Moreover, we found the positive correlation of MVP expression with SI (Doc) and SI (Doc + Gem) in undifferentiated pleomorphic sarcoma samples from patients treated with neoadjuvant chemotherapy (Table 5). In synovial sarcomas we demonstrated positive correlation of ABCC1 expression with SI (Ifo) and SI (Dox + Ifo) as well as ABCG2 expression gene with SI (Doс). We did not analyze the groups of primary and relapsed patients because of insufficient groups size.

Expression of P-Glycoprotein and ABCG2 in Primary Cultures of STS
We analyzed the protein Pgp and ABCG2 expression in primary STS cultures of 1-2 passages by flow cytometry. We used cell cultures K562/i-S9 with Pgp overexpression and HBL-100/Dox with ABCG2 overexpression as positive controls.
Pgp expression was evaluated in 44 samples. The threshold value was 5% of cells population, which expressed Pgp. We supposed, it was the minimal level of protein expression with significant contribution in the resistance of tumor population and with the effect on clonal selection under the drug exposure. In 5 samples (11%) the Pgp expression was above this value with mean expression level 19.4% of STS cell population in positive group ( Figure 3). As in this sample series the amount of medium and high Pgp expression were lower than expected, we selected 8 STS samples with different Pgp expression and analyzed the protein amount by Western blotting (Figure 4). The results of flow cytometry were confirmed by Western blot analysis that supported the observation on rare Pgp expression in studied sample series.  Pgp and ABCG2 expressions were not associated with such clinical patient characteristics as sex, age, disease stage, degree of malignancy, chemo/radiotherapy delivery and the treatment response (Table 6). ABCG2 expression did not vary in different histological STS subtypes. Within the subgroups we did not find the association of ABCG2 with SI of primary STS cultures.
We demonstrated that Pgp expression frequency in synovial sarcomas was significantly higher than in undifferentiated pleomorphic sarcomas (p = 0.04). In synovial sarcomas 5 out of 6 samples were obtained from the relapsed patient, and in this subgroup, we showed strong positive correlation of Pgp expression with SI(Gem) (r = 0.97, p = 0.005). However, it should be mentioned that Gem is not Pgp substrate. ABCG2 expression was analyzed in 39 samples, the threshold value was 1% as ABCG2 expresses in cancer stem cells being the minor population. We found 17 samples (44%) with the expression level above this threshold with mean expression level 3,75% of STS cell population in positive group ( Figure 5). Thus, we did not find correlations in ABCB1 and ABCG2 mRNA expression and Pgp and ABCG2 protein expression, respectively.
Pgp and ABCG2 expressions were not associated with such clinical patient characteristics as sex, age, disease stage, degree of malignancy, chemo/radiotherapy delivery and the treatment response (Table 6). ABCG2 expression did not vary in different histological STS subtypes. Within the subgroups we did not find the association of ABCG2 with SI of primary STS cultures.
We demonstrated that Pgp expression frequency in synovial sarcomas was significantly higher than in undifferentiated pleomorphic sarcomas (p = 0.04). In synovial sarcomas 5 out of 6 samples were obtained from the relapsed patient, and in this subgroup, we showed strong positive correlation of Pgp expression with SI (Gem) (r = 0.97, p = 0.005). However, it should be mentioned that Gem is not Pgp substrate.

Identification of Mutations in Several Genes of ABC-Transporter Family
We analyzed the mutations in several genes from ABC-transporter family in the group of 39 STS samples (synovial and pleomorphic histology subtypes). We selected 36 genes associated with tumor progression out of total 300 genes from ABC-transporters family. We found mutations in 9 out of 39 samples, in 3 samples (STS 29, 104, 119) we observed 2-3 mutations. In the Table 7, A3, A13, C1, C6, C11, B1, and G2 genes are associated with drug elimination from cell, whereas A1 and B11 genes play a role in transport of lipids and biliary acids. Mutations were found in the most significant MDR-associated genes B1, C1, G2 in 1, 2, and 1 cases, respectively (10% of total number of samples).

Discussion
We hypothesized that expression of ABC-transporters is the important mechanism of MDR in STS as it was shown for malignant peripheral nerve sheath tumor and sarcomas of the pulmonary artery [16,18]. Further, it was described earlier that ABCC1/MRP1 expression may have a prognostic significance for patients with STS of high risk, who received anthracycline-based treatment [19]. In cases of embryonal rhabdomyosarcoma ABCC1 expression correlated with poor survival [20]. More frequently this disease is associated with ABCG2 expression [21].
However, in our study we found only 11% of samples with Pgp expression, and only in 1 case we observed 30% Pgp-positive cells. The similar results were obtained for ABCG2 expression: This index reached 16% in rare cases and generally did not exceed 2-5%. As the low level of Pgp and ABCG2 expression in cancer cells resistant to the studied drugs was observed, we propose that other mechanisms of MDR may be more important in STS. Possibly, the rare Pgp expression is associated with the histological tumor subtypes in our specific sample series as in literature high Pgp expression was described in rhabdomyosarcomas [17] and low expression of this gene was shown to be associated with Ewing sarcoma [22]. Interesting, results were obtained for the subgroup of 6 samples of synovial sarcoma, 5 of which belonged to relapsed patients. We demonstrated the increased Pgp expression strongly positively correlated with the resistance to Gem, nevertheless, Gem is not the Pgp substrate. However, one study described the increased sensitivity to Gem in Pgp or MRP1-overexpressing cell lines and their counterparts without MDR phenotype [23]. In our study, we suppose, that Pgp expression does not lead to Gem resistance, but Gem may activate Pgp expressions [24].
In primary analysis of the whole sample series, we did not observe any strong correlations of MDR-associated genes expression and resistance of primary cell cultures. Moreover, in the analysis of undifferentiated pleomorphic sarcoma subgroup we demonstrated the negative correlation for the ABCB1 and ABCG2 expression and SI (Doc) or SI (Gem + Doc). As corresponding proteins were not expressed on the sufficient level in STS cells in our sample series, we guess that decreased expression of ABCB1 and ABCG2 in resistant cells is the indicator of the activation of the specific signaling resulting in STS resistance. If we consider other MDR mechanisms in undifferentiated pleomorphic sarcoma, then it may be the activation of LAPTM4A and LAPTM4B, which are responsible for the transport of small molecules through endosomal and lysosomal membranes. Its increased expression correlated with lower chemosensitivity, especially to the anthracycline-based therapy [25].
We found that studied ABC-transporter genes correlated with each other by mRNA expression level; however, we did not observe sufficient protein amount in spite of high level of mRNA expression in most samples. We believe that it could be associated with the alternative regulation on translation level. It is known that small non-coding RNA (miRNA) participate in posttranscriptional regulation of gene expression either via activation of mRNA degradation or via inhibition of translation repression [26,27]. Thus, canonic mRNA binding with complementary seed regions in 3 UTR mRNA results in translation repression, and, similarly, regulatory RNA-binding proteins change its stability or further translation process [28] 38. In one study it was shown that the three-component complex of HuR, miR-19b, and UTR inhibits the expression of ABCB1/Pgp [29]. In another study it was demonstrated that interactions of ABCB1-3 -UTR-miR-485-3p and ABCC2-3 -UTR-miR-26a-5p induce the translation repression of Pgp and MRP1, which was resulted in the low expression on protein level in spite of the high mRNA expression observed [30].
In our study we demonstrated that resistance to Doc and its combination with Gem was higher in patients with good response to the therapy Dox + Ifo. It could prove the hypothesis that tumor cells survived the neoadjuvant chemotherapy and undergone the clonal selection, obtain higher resistance to Doc. In addition, it could be in accordance with the data that viability of tumor cells with low-grade therapy-related pathomorphism is more dependent from the tumor microenvironment [31]. After culturing in vitro this protective property was withdrawn, and cells demonstrated the chemosensitivity in MTT-test.
We found that the expression of MVP (LRP) gene on mRNA level is lower in tumor samples from the relapsed patients than in primary tumors. It could be associated with the differentiation grade in primary and relapsed tumors. In one study it was demonstrated that MVP protein was predominantly expressed in differentiated cells in rhabdomyosarcoma samples before and after chemotherapy, possibly MVP expression allow rhabdomyosar-coma cells to survive the chemotherapy [32]. In analysis of undifferentiated pleomorphic sarcoma subgroup, we found the positive correlation in MVP expression and resistance to Doc and Doc + Gem combination, and this correlation become stronger in the cells obtained from patients after neoadjuvant chemotherapy. That was in agreement with our previous study on glioblastoma samples. We showed that increased MVP expression on mRNA level was associated with the low proliferation and high resistance to Temozolomide [33]. In the undifferentiated pleomorphic sarcoma subgroup, we proposed direct action of MVP as an MDR protein, but this conclusion requires further verification on a larger group of patients and study of MVP at the protein level.
Our study had a number of limitations: Some of the analyzed groups had very few samples. Almost the entire group of synovial sarcomas with increased Pgp expression came from relapsed patients, and we cannot establish whether high Pgp expression is characteristic only of relapse samples or of all synovial sarcomas. The next limitation is that at this stage of investigation, we studied the expression of YB-1, ABCC1, and MVP only at the mRNA level, but not at the protein level, which could provide new information concerning their involvement in the MDR formation of STS.

Methodological Approaches
We assessed the expression of ABC-transporters, MVP and YB-1 and analyzed their correlation with chemosensitivity of corresponding STS cells to the above-mentioned chemotherapeutic agents. Drug resistance was estimated using in vitro chemosensitivity assay proposed by Kurbacher et al. [34], which, currently, are widely used in many modifications [35][36][37][38], based on different methods for estimation of cell viability [39][40][41].

Primary Cancer Cell Cultures and Chemosensitivity Assay
Tumor chemosensitivity assay was performed as a routine procedure immediately following surgery. Solid tumors were obtained during surgery and cut into smaller fragments (1 mm 3 ), which were then dissociated by incubation in 5-10 mL sterile collagenase mix for 2-3 h at 37 • C on a shaker to prepare suspensions of single cells. One part of the cells was used for the chemosensitivity test, and the other one for the analysis of multi-drug resistance. After adjusting the concentration of cells in the suspension to 1-2 × 10 5 cells/mL, 100-µL cell suspensions were added to each well of a 96-well microplate. Single agents were tested at six different concentrations of a standard test drug concentration (TDC), in particular, 6.25, 12.5, 25, 50, 100, and 200% of the peak plasma concentration of the drug (Table 8), as it was proposed by Andriotti et al. [42]. The TDCs were based on pharmacokinetic data for standard doses of the agents, adjusted to give good discrimination [42]. Plates were incubated for 5-6 days at 37 • C with 95% humidity in a 5% CO 2 incubator. Cell viability was measurement using resazurinbased assay described previously [25]. The results in vitro chemosensitivity tests were interpreted and compared using the sensitivity index SI (SI = 600-sum of % inhibition at 200, 100, 50, 25, 12.5, and 6.25% TDC) [42][43][44].

Cytology for Isolated Cell Culture
In order to determine the percent of malignant cells for each isolated primary culture, cytological study was performed. Derived cells were used to thin-layer slides preparation through a cytocentrifugation process by the Thermo Shandon Cytospin 3. Morphological assay to determine the percent of malignant cells in Leishman-stained slides were performed on a microscope «Nikon Eclipse Ci-S» (Nikon Corporation, Tokyo, Japan) at 1000× magnification in 3 fields of view.

Quantitative PCR (Q-PCR)
Total RNA was isolated with PureZOL RNA Isolation Reagent (BIO-RAD, Hercules, CA, USA) according to the manufacturer's protocol. The RNA quality was checked by electrophoresis in 1% agarose gel containing 0.01% ethidium bromide. Samples with clearly visible 18S and 28S RNA bands were used for further analysis. For the synthesis of cDNA, we used a set of reagents for reverse transcription with primers Random6 (Syntol, Moscow, Russia). The real-time PCR reaction was performed using the intercalating fluorescent agent Eva Green (Synthol, Moscow, Russia) and Taq

Exome Capture, Alignments and Base-Calling
Exomes for the 39 patients were captured with Agilent SureSelect Focused Exome. Libraries were indexed, pooled, and sequenced on Illumina HiSeq2000 machines (paired-end, 250-bp reads). Reads were mapped to GRCh38/hg38 build using BWA 0.7.15 [47] followed by marking duplicate reads with Picard-tools 2.20 (Picard-tools 2.20. Available online http://broadinstitute.github.io/picard/ (accessed on 14 September 2020)). GATK4 [48] base quality score recalibration pre-processing step was performed to detect systematic errors made by the sequencing machine. Tumor-only variant calling was performed on tumor samples with no paired normal using advantages of normal cell contamination implemented in GATK4 Mutect2 (tumor-only mode) probabilistic models for genotyping and filtering.

Statistical Analysis
Data were presented as M ± S.D. The Mann-Whitney test was carried out to analyze the differences between groups and the Spearman's correlation coefficient was calculated to quantitate the degree of correlation between parameters. The Kruskal-Wallis test with post test (compare all pairs of columns) was used to analyze groups with different histology types. GraphPad Prism 6.0 was used (GraphPad Software, San Diego, CA, USA). The difference was considered statistically significant at p-value < 0.05 (two-tailed).

Conclusions
Overall, we used STS primary cultures as a new approach for the study of MDR mechanisms in STS of different types. Pgp protein expression is a very rare event and probably does not have clinical significance, with the exception of synovial sarcoma. Our results afford us to conclude that MVP expression may play role in MDR of undifferentiated pleomorphic STS. Mechanisms of resistance in STS require further study and search for new targets.  Institutional Review Board Statement: All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of N.N. Blokhin NMRCO (protocol code 18-29-09095 and date of approval 28 September 2018).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patients to publish this paper.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy restrictions.

Conflicts of Interest:
The authors declare no conflict of interest.