Effects of a Novel Thiadiazole Derivative with High Anticancer Activity on Cancer Cell Immunogenic Markers: Mismatch Repair System, PD-L1 Expression, and Tumor Mutation Burden

Microsatellite instability (MSI), tumor mutation burden (TMB), and programmed cell death ligand-1 (PD-L1) are particularly known as immunotherapy predictive biomarkers. MSI and TMB are closely related to DNA mismatch repair (MMR) pathway functionality, while the PD-L1 checkpoint mediates cancer cell evasion from immune surveillance via the PD-L1/PD-1 axis. Among all the novel triazolo[3,4-b]thiadiazole derivatives, the compound KA39 emerged as the most potent anticancer agent. In the present study, potential alterations in MSI, TMB, and/or PD-L1 expression upon cell treatment with KA39 are explored. We tested three MMR-deficient (DLD-1, LS174T, and DU-145) and two MMR-proficient (HT-29 and PC-3) human cancer cell lines. Our findings support KA39-induced PD-L1 overexpression in all cancer cell lines, although the most outstanding increase was observed in MMR-proficient HT-29 cells. MSI analysis showed that KA39 affects the MMR system, impairing its recognition or repair activity, particularly in MMR-deficient DLD-1 and DU-145 cells, enhancing oligonucleotide production. There were no remarkable alterations in the TMB between untreated and treated cells, indicating that KA39 does not belong to mutagenic agents. Taking together the significant in vitro anticancer activity with PD-L1 upregulation and MSI increase, KA39 should be investigated further for its implication in chemo-immunotherapy of cancer.


Cell Lines and Culture Conditions
Our study was conducted in five well-established human cancer cell lines: three colorectal adenocarcinoma (DLD-1, HT-29, and LS174T) and two prostate (PC-3 and DU-145) ( Table 1). All cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultivated in different culture media according to supplier instructions. All growth media were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All cancer cell lines were cultured as monolayers and maintained at 37 • C in a humidified 5% CO 2 atmosphere.

Cell Treatment
Among several newly synthesized triazolo [3,4-b]thiadiazole derivatives that have been tested for anticancer activity, KA39 has been shown as the most potent, inducing significantly higher cytostatic and cytotoxic activity against all tested cancer cell lines. In addition to KA39, two triazolo [3,4-b]thiadiazoles derivatives with significant structural similarity (>95%), XK71 and XK57, were also tested. All molecules were synthesized according to procedures that we previously reported (Figure 1; Figure S21) [4,5].

In Vitro Antiproliferative Activity
The in vitro anticancer activity of KA39 against all cancer cell lines was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide quantitative colorimetric MTT assay, as previously reported [4,5]. Briefly, cells were seeded into a 96-well plate at a density of 8 × 10 3 cells per well and maintained for 72 h. After 24 h of cell growth, cells were treated with KA39 in concentrations of 1-100 µM. Following 48 h of drug exposure, 50 µL of MTT (5 mg/mL) was added to each well and cells were incubated for 3 h. The absorbance of the converted dye was recorded at a wavelength of 540 nm on an ELISA reader (Versamax, Orleans, LA, USA).
MTT assay provides the required absorption values for determining three doseresponse parameters, GI 50 , TGI, and IC 50 , using the linear progression method. All the experiments were carried out in triplicate. GI 50 and TGI are drug concentrations that signify the cytostatic effect of a tested compound and induce 50% and 100% of cell growth inhibition, respectively; IC 50 is the drug concentration that implies the cytotoxic effect of a tested compound and leads to a 50% decrease in cell viability [22,23]. The three parameters were determined using the mean of cell survival in all nine absorbance measurements, including control 24 h (Ct24), control 72 h (Ct72), and the seven drug concentrations (Tt72). The percentage of growth inhibition was calculated as [(Tt72x) − (Ct24)/(Ct72) − (Ct24)] × 100 for concentrations for which Tt72x > Ct24 and [(Tt72x) − (Ct24)/Ct24] × 100 for concentrations for which Tt72x < Ct24. GI 50

Flow Cytometric Analysis of Surface PD-L1 Expression
The impact of KA39 on PD-L1 expression was studied in four cancer cell lines, two colorectal (DLD-1 and HT-29) and two prostate (PC-3 and DU-145). Cells were seeded in a 6well plate at a density of 5 × 10 5 cells/well and maintained for 24 h at 37 • C in a humidified 5% CO 2 atmosphere. After 24 h of cell growth, the culture medium was replaced with fresh medium; each cell line was treated with KA39 according to the defined values of the TGI concentration and IC 50 (µM), while untreated cells served as controls ( Figure 1; Table 1). Cells treated at the TGI concentration (µM) were allowed to grow for 72 h in contrast to IC 50 (µM), in which cells were exposed for 48 and 72 h. Following drug treatment, cells were collected after being washed with ice-cold PBS (pH 7.4) (Sigma-Aldrich, St. Louis, MO, USA) and detached enzymatically with standard trypsinization. All centrifugations, performed at 1500 rpm for 5 min, included medium discard and washing steps with 2 mL of cold cell-staining buffer (BioLegend, San Diego, CA, USA). Subsequently, 25 × 10 4 of cells were resuspended in 100 µL of cell-staining buffer and then stained with 5 µL of anti-PD-L1 antibody (PE/Cy7 anti-human CD274, Biolegend, San Diego, CA, USA). Cells were incubated at room temperature in the dark for 15 min and then resuspended with 800 µL of cell-staining buffer. The expression of PD-L1 was analyzed on a flow cytometer (CyFlow ® , SL, Partec, GmbH, Germany) using Partec Flomax software version 2.3 (Münster, Germany). For each sample, flow cytometric analysis was carried out in triplicate. The absolute values of the KA39-induced alterations in PD-L1 expression were reduced to a percentage, and controls (untreated cells) were defined as 100% of PD-L1 expression.

DNA Extraction
DNA extraction was carried out in frozen cell pellets using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). According to manufacturer's instructions, 200 µL of ATL buffer and 21 µL of proteinase K were added and then cell samples were incubated at 56 • C for 1 h. Following further incubation at 90 • C for 15 min, 200 µL of AL buffer as well as 200 µL of 100% ethanol were added. After brief vortex mixing, the DNA extract was transferred into a QIAamp Mini spin column (in a 2 mL collection tube) and centrifuged at 1200 rpm for 1 min. Subsequently, the QIAmp Mini spin column was placed into a new 2 mL collection tube and 500 µL of AW1 buffer was added. Once samples were centrifuged at 8000 rpm for 1 min, the QIAmp Mini spin column was placed into a new 2 mL collection tube and 500 µL of AW2 buffer was added. Like before, samples were centrifuged at 8000 rpm for 1 min. Afterward, the QIAamp Mini spin column was placed into a new 2 mL collection tube and centrifugation was carried out at full speed (14,000 rpm) for 3 min. The purified DNA was eluted by adding the appropriate volume of AE buffer (30-50 µL), and samples were finally centrifuged at 14,000 rpm for 1 min. The DNA concentration (ng/µL) of all samples was determined spectrophotometrically (NanoDrop2000, Thermo Fisher Scientific, Waltham, MA, USA).

MSI Fragment Analysis
Alterations in the MSI status were studied in three colorectal (DLD-1, HT-29, and LS174T) and two prostate (PC-3 and DU-145) human cancer cell lines upon treatment with KA39. Briefly, cells were seeded in a 6-well plate at a density of 3 × 10 5 cells/well and cultured for 24 h at 37 • C in a humidified 5% CO 2 atmosphere. After 24 h of cell growth, the culture medium was replaced by fresh medium and cells were subsequently treated with KA39 at the TGI concentration and IC 50 (µM) for 48 h (Figure 1; Table 1). Following 48 h of drug exposure, the culture medium was discarded and cells were collected after being washed twice with ice-cold PBS (pH 7.4) (Sigma-Aldrich, St. Louis, MO, USA) and detached enzymatically with standard trypsinization. Afterward, cells were centrifuged twice at 2000 rpm for 5 min, while one further centrifugation was carried out in a microcentrifuge at 3000 rpm for 15 min. DNA was extracted and then subjected to multiplex PCR with fluorescently labeled primers obtained from Thermo Fisher Scientific (Waltham, MA, USA) [24] (Table 3). PCR was carried out using the commercial QIAGEN Multiplex PCR Kit (Qiagen, Hilden, Germany). Furthermore, MSI analysis was performed according to the NCI panel (Bethesda panel), which consists of two mononucleotide (BAT25 and BAT26) and three dinucleotide (D5S346, D17S250, and D2S123) repeats [25]. Following the manufacturer's recommendations, each 20 µL of the PCR multiplex reaction mix was composed of the following: 12.5 µL of 2× Qiagen multiplex PCR master mix (HotStarTaq DNA Polymerase (5 units/µL), 6 mM MgCl 2 , pH (8.7), and dNTP mix (dATP, dCTP, dGTP, dTTP)), 2.5 µL of 10× MSI primer mix, template DNA whose volume was variable depending on the concentration of DNA isolated (ng/µL), and RNase-free water, which was added to reach a total volume of 20 µL. In the negative control, DNA was replaced by water. The PCR amplification program started with an activation step of HotStarTaq DNA Polymerase at 95 • C for 15 min, followed by 40 cycles of 30 s at 95 • C (denaturation), 90 s at 51 • C (annealing), and 60 s at 72 • C (extension), and a final extension step of 30 min at 60 • C (Veriti™ 96-Well Thermal Cycler; Thermo Fisher Scientific, Waltham, MA, USA). To ascertain whether the amplification of the expected amplicons was successful, PCR products were analyzed by capillary electrophoresis. More specifically, the PCR products, stained with ethidium bromide, were loaded on 2% agarose gel in 1× TAE buffer, run at 97 V for 45 min (electrophoresis system; Bio-Rad Laboratories, Hercules, CA, USA) and finally visualized using the MiniBis Pro (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel). For fragment analysis, the PCR products were denatured by adding 14.5 µL of a mixture composed of GeneScan™ 500 LIZ™ Size Standard (Thermo Fisher Scientific, Waltham, MA, USA) and Hi-Di™ Formamide (Thermo Fisher Scientific, Waltham, MA, USA) to 1 µL of each diluted PCR product. To achieve DNA denaturation, samples were incubated at 95 • C for 3 min (Veriti™ 96-Well Thermal Cycler; Thermo Fisher Scientific, Waltham, MA, USA) and subsequently cooled down in the freezer for 3 min. DNA fragments were analyzed on the GeneScan 500 LIZ Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA), and data analysis was conducted using GeneMapper 4.0 software version 4.0 (Applied Biosystems, Foster City, CA, USA). MSI analysis was conducted by quantifying all DNA fragments per nanogram of DNA input in PCR.

Statistical Analysis
Student's t-test was used to compare the level of significance between the experimental groups. Differences with a p-value less than 0.05 were considered statistically significant. Microsoft Excel version 2010 (Microsoft Hellas, Athens, Greece) was used.

MSI, PD-L1 Expression, and TMB Analysis in Human Colorectal and Prostate Cancer Cells upon Treatment with KA39
KA39 induced the most significant cytostatic and cytotoxic effects on the five tested human cancer cell lines (Table 1). However, XK71 and XK57 displayed low in vitro anticancer activity in the tested cancer cell lines, and no significant alterations in MSI and PD-L1 expression were impelled (p > 0.01) The alignment of cancer cell sensitivity to KA39 was DLD-1 > PC-3 > DU-145 > HT-29 > LS174T, with DLD-1 cells being more and LS174T less sensitive (p < 0.01). All results concerning the in vitro anticancer activity and changes in MSI and PD-L1 expression induced by XK71 and XK57 are provided as Supplementary Materials (Table S21-S23; Figures S22 and S23).
Among all human cancer cell lines included in our experimental studies, DLD-1, DU-145, and PC-3 cells were the most sensitive to KA39, as TGI and IC 50 values indicate (Table 1). Nevertheless, HT-29 cancer cells, the cell line less susceptible to KA39, exhibited the most outstanding increase in PD-L1 expression levels in comparison with DLD-1, DU-145, and PC-3 cells. However, MSI appeared to be significantly enhanced in DLD-1 and DU-145 cancer cells in which KA39 generated a higher cytostatic and cytotoxic effect. Comparing with DLD-1 and DU-145 cells, less meaningful but statistically significant MSI enhancement was detected in HT-29, LS174T, and PC-3 cells. With reference to TMB assay and non-synonymous mutations, no considerable alterations were displayed by the tested cancer cell lines, as shown by the TMB values of untreated and treated cancer cells.

Discussion
PD-L1, also termed as CD274 or B7-H1, is the physiological ligand of the PD-1 receptor, and both are most important immune checkpoints. PD-L1 expressed by tumor cells is bound to PD-1, which is located on activated T cells. Cancer cells, through PD-L1/PD-1 interaction, are capable of suppressing the tumor-reactive T cells and evading cancer immune surveillance [26,27]. Even though chemotherapy provides important therapeutic benefits, harmful effects may be induced on anticancer immunity, such as the drug-induced PD-L1 expression in cancer cells. To the best of our knowledge, current studies are addressing the effects of anticancer agents on PD-L1 expression in a variety of cancers, including pancreatic, ovarian, and breast [27,28]. Moreover, there appears to be a dose-dependent relationship between PD-L1 upregulation and chemopreventive or cytotoxic agents as, for example, in decitabine and cisplatin administered in leukemia and hepatoma cells, respectively [28,29]. Qin et al. [29] showed that PD-L1 is overexpressed in H22 hepatoma cells following exposure to cisplatin at a concentration less than IC 50 . According to our findings, all four cancer cell lines treated with KA39 showed significantly elevated PD-L1 expression levels, though the most impressive increase was induced in HT-29 cancer cells (Figure 2A-D). HT-29 cells were less sensitive to KA39 with the TGI concentration and IC 50 being approximately 2-fold higher compared to those of DLD-1, DU-145, and PC-3 cells (Table 1). PD-L1 overexpression in the treated HT-29 cells occurred in a dose-dependent manner.
It is suggested that oncogenic signaling pathways such as RAS-RAF-MEK-ERK, PI3K-AKT-mTOR, JAK-STAT, and NF-κB are strongly involved in the anticancer agent-mediated PD-L1 expression probably by a signaling crosstalk. In view of the PI3K-AKT-mTOR pathway, inhibition of either PI3K or its downstream signaling molecule AKT leads to repression of PD-L1 expression in tumor cells [30][31][32]. On the contrary, as shown in experimental studies using breast cancer cell lines, loss of phosphatase and tensin homolog (PTEN), due to mutation or depletion, leads to PI3K activation, which, in turn, upregulates the expression of PD-L1 [31,32]. Regarding the PTEN-deficient PC-3 cell line [21], KA39-induced PD-L1 expression was barely higher than in PTEN wild-type DU-145 cells; however, it was not as notable as in HT-29 and DLD-1 cells (both PTEN wild type), in which PD-L1 was upregulated to a far greater extent (Figure 2A,B). Moreover, taking into consideration that the KA39 derivative behaves as an AKT inhibitor [6], downregulation of PD-L1 would be expected. Nevertheless, our results support an acceleration of PD-L1 expression in response to KA39 exposure irrespective of the PTEN status and AKT inhibition, signifying a distinct mechanism of action.
Accumulating evidence suggests that PD-L1 upregulation may occur in the context of DNA-damage-induced signaling in tumor cells [33]. As we have previously reported, KA39 induces topIIα inhibition as well [5]. TopIIα inhibitors, as DNA-damaging agents, stimulate the expression of type I IFNs and other cytokines via the activation of the cGAS-STING-TBK1-IRF3 pathway [34]. Furthermore, an association of the cGAS-STING pathway with PD-L1 upregulation has been reported [35]. Wang et al. [34] also found an immune response impelled by the topoisomerase II inhibitor, in particular teniposide-induced PD-L1 expression in multiple cancer cells in vitro. Mechanically, the cytoplasmic DNA, caused by exposure to topII inhibitors, serves as a DNA damage signal, which can be sensed by cyclic GMP-AMP synthase (cGAS). cGAS binding to cytosolic DNA promotes the synthesis of the second messenger cyclic GMP-AMP (cGAMP), which binds to the adaptor protein Stimulator of IFN Gene (STING). The activated STING recruits a signaling cascade that triggers the transcription of type I IFNs genes [36]. It is thought that type I INFs regulate PD-L1 expression through the JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis, with IRF1 being bound to the PD-L1 promoter and hence triggering its production [33,34,37].
The MSI phenomenon emerges from a deficient mismatch repair (dMMR) mechanism, which is responsible for the accumulation of mutations in the genome's short tandemly repeats (STRs), termed as microsatellites [9]. There are three distinct types of MSI phenotypes linked to MMR functionality: (1) MSI-H associated with a dMMR system, (2) low microsatellite instability (MSI-L), and (3) MSS, with the last two being related to pMMR status [38]. The MSI-H phenotype appears in several sporadic cancers, including colorectal, gastric, small intestine, urothelial, endometrial, and, more rarely, prostate [9,39]. Loss of MMR function contributes to enhanced chemoresistance as dMMR cells are less sensitive to commonly used chemotherapeutic drugs such as alkylating agents, platinum compounds, topoisomerase poisons, and purine analogues [40]. However, anti-PD-1 and anti-PD-L1 immunotherapies are much more efficient in dMMR/MSI-H tumors. Concurrent coexistence of neoantigens produced by MMR mutations, with PD-L1 immune checkpoint expression, provides a breeding ground for MSI-H tumors to be treated with anti-PD-L1 therapy [38,[41][42][43].
The MSI condition is linked to an impaired MMR mechanism whose dysfunctionality results from mutations, either germline or spontaneous, in MMR genes [12]. The MMR pathway, responsible for the maintenance of genomic integrity, undertakes the repair of postreplicative DNA base mismatches as well as inserted/deleted loops incorporated into microsatellites. Four key genes support MMR's functionality: mutL homologue 1 (MLH1), postmeiotic segregation increased 2 (PMS2), mutS homologue 2 (MSH2), and mutS6 (MSH6) [44]. The MMR mechanism is initiated with the mismatch being recognized by the MutSα heterodimer (MSH2/MSH6). Afterward, MutSα interacts with the MutLα complex (MLH1/PMS2), forming a "sliding clamp" that moves up and down on the DNA sequence that contains the mismatch. This sliding clamp acts in an ATP-hydrolysisdependent manner [45]. Once the daughter strand has been identified, the MutSα/MutLa complex guides DNA Exonuclease 1 (EXO1) in order to carry out an excision at the mismatch site. The repair process is completed once the removed DNA sequence is replaced by DNA polymerase δ and the remaining gaps are sealed by DNA ligase I [46,47]. Microsatellites are known for their extreme vulnerability to errors that arise from DNA replication, recombination, or external sources such as radiation or chemical agents [44].
Our study points out a considerable accumulation of oligonucleotides in all five cancer cell lines treated with the anticancer agent KA39, comprising dMMR as well as pMMR cancer cells. Nonetheless, augmentation of DNA fragments, impelled by KA39, was remarkably higher in dMMR DLD-1 and DU-145 cells than in pMMR HT-29 and PC-3 cells, even in dMMR LS174T cells. Furthermore, it is noteworthy that MSI was enhanced in terms of quantity, as more oligonucleotides resulted from KA39 exposure, whereas no alterations were revealed in the relocation of DNA fragments. Each MSI-H cancer cell line carries specific MMR deficiencies (Table 2). DLD-1 cells are dMMR cancer cells bearing a missense mutation in the MSH6 gene, which is responsible for complete loss of the protein. In the absence of the MSH6 protein, single-base mismatches cannot be repaired, though the MMR pathway retains its functionality in general terms. The lack of MSH6 expression does not influence the other MutS proteins, as MSH2 counterbalances its loss by binding to MSH3 [48]. Thus, it can be suggested that in DLD-1 cells, the MMR pathway maintains its recognition activity to a certain extent. With respect to dMMR DU-145 cells, the MutLα heterodimer is completely absent, as both MLH1 and PMS2 proteins are lacking, and consequently the MMR repair activity is significantly diminished. It is interesting to note that the loss of the MLH1 protein has a crucial impact on the MMR mechanism as it leads to inactivation of MutLα's endonuclease activity, degradation of PMS2, and, hence, an extreme accumulation of errors [17,18,49]. With regard to dMMR LS174T cells, all proteins (MSH2, MSH6, PMS2, and MLH1) are expressed at low levels. However, studies have shown that MLH1 appears to a far lesser extent, probably indicating an attenuated activity of the MutLa complex [50]. Presumably, the KA39 agent further deteriorated the MMR mechanism, mainly in dMMR cells, by disrupting the recognition or repair activity of the MMR mechanism. Nevertheless, the exact molecular mechanism through which KA39 disrupts the MMR pathway is unclear. MSI-H cancer cell lines such as DLD-1 (MSH6 deficiency) and DU-145 (MLH1 and PMS2 deficiencies) cells showed a greater susceptibility to KA39 than the MSS cancer cell line HT-29, pointing out a kind of synthetic lethality. However, the genetic and molecular profile of a cell line influence its cellular response to an anticancer drug; MSS PC-3 and MSI-H LS174T cells share similar sensitivity to KA39 as MSI-H DU-145 and MSS HT-29 cells, respectively (Table 1).
Studies related to the MMR pathway support an association of MMR deficiency with resistance to topII inhibitors, though the findings around this area are controversial [51,52]. In pMMR cancer cells, the cleavage complex, induced by a topII inhibitor, is recognized by the MMR pathway, activating eventually the apoptotic procedure. In contrast, the genomic instability of dMMR cells increases the mutation rates in the topII gene, leading, as a consequence, to mutated topII unable to be bound to a topII inhibitor [53,54]. As previously discussed, our results regarding the drug sensitivity to KA39 exhibit heterogeneity, which may be attributable to their specific characteristics.
A growing body of evidence supports the implication of AKT in the MMR pathway via PMS2, a component of the MutLα heterodimer. More particularly, binding of the phosphorylated AKT Ser473 to PMS2 leads to the degradation of MutLα's component, inducing genomic instability and DNA damage. By contrast, inhibition of AKT ensures the stability and nuclear localization of PMS2. It appears that cells with hyperactivated AKT carry ineffective MMR responsible for enhanced accumulation of mutations, drug resistance, and inactivation of the apoptotic procedure [55,56]. Even though KA39 acts as a potent AKT inhibitor as well [6], the MMR pathway was significantly impaired, pointing out that KA39 is not a pure AKT inhibitor. Probably, KA39 targets other elements of the repair mechanism.
The TMB represents the number of exonic non-synonymous mutations per megabase (Mb) [57]. Given that the TMB serves as a measure of somatic coding mutations, the volume of neoantigens can be evaluated and, by extension, the responsiveness to ICB therapy can be predicted. It is interesting to note that a higher number of mutations signifies higher response rates, as shown in MSI-H tumors known for their immunogenicity, which is associated with an increasing TMB [42,58]. KA39 produced no direct alterations on the TMB and on the non-synonymous mutation number in the tested cancer cell lines independent of their MMR status. However, a significant increase (>2-fold) of unknown importance was induced at the synonymous mutation number in DLD-1 cells, which bear an MSH6 deficiency (Table 6).

Conclusions
The impact of the KA39 triazolo [3,4-b]thiadiazole derivative was investigated on three predictive biomarkers for cancer immunotherapy: PD-L1, MSI, and TMB. According to our findings, a notable increase in PD-L1 expression and MSI was demonstrated, presumably in the context of DNA damage introduced by KA39, while no changes in the TMB were induced. Conclusively, KA39 cannot be considered a mutagenic agent, and microsatellite errors were accumulated by destabilizing the MMR mechanism during cancer cell treatment. Altogether, the significant in vitro anticancer activity in combination with PD-L1 upregulation and MSI enhancement implies that KA39 is a promising anticancer agent able to be developed for cancer chemo-immunotherapy.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/pharmaceutics13060885/s1, Figures S1-S20: Flow cytometric analysis data, Figure S21: Chemical structures of XK57 and XK71, Figure S22: MSI alterations induced by XK57 derivative in five human cancer cell lines, Figure S23: MSI alterations induced by XK71 derivative in five human cancer cell lines, Tables S1-S20: Flow cytometric analysis data-Regions analysis, Table S21: Cytostatic and cytotoxic effects induced by XK57 and XK71 on the tested human cancer cell lines,