To study the molecular mechanisms responsible for inducing the spatial proliferation of malignant melanomas (MM), three-dimension (3D) spheroids were produced from several MM cell lines including SK-mel-24, MM418, A375, WM266-4, and SM2-1, and their 3D architectures and cellular metabolisms were evaluated by phase-contrast microscopy and Seahorse bio-analyzer, respectively. Several transformed horizontal configurations were observed within most of these 3D spheroids, and the degree of their deformity was increased in the order: WM266-4, SM2-1, A375, MM418, and SK-mel-24. An increased maximal respiration and a decreased glycolytic capacity were observed within the lesser deformed two MM cell lines, WM266-4 and SM2-1, as compared with the most deformed ones. Among these MM cell lines, two distinct cell lines, WM266-4 and SK-mel-24, whose 3D appearances were the closest and farthest, respectively, from being horizontally circular-shaped, were subjected to RNA sequence analyses. Bioinformatic analyses of the differentially expressed genes (DEGs) identified KRAS and SOX2 as potential master regulatory genes for inducing these diverse 3D configurations between WM266-4 and SK-mel-24. The knockdown of both factors altered the morphological and functional characteristics of the SK-mel-24 cells, and in fact, their horizontal deformity was significantly reduced. A qPCR analysis indicated that the levels of several oncogenic signaling related factors, including KRAS and SOX2, PCG1α, extracellular matrixes (ECMs), and ZO1 had fluctuated among the five MM cell lines. In addition, and quite interestingly, the dabrafenib and trametinib resistant A375 (A375DT) cells formed globe shaped 3D spheroids and showed different profiles in cellular metabolism while the mRNA expression of these molecules that were tested as above were different compared with A375 cells. These current findings suggest that 3D spheroid configuration has the potential for serving as an indicator of the pathophysiological activities associated with MM. A malignant melanoma (MM) originates from melanocytes, in which a photoprotective pigment, melanin is synthesized, and thus can be found in various organs that contain cells that produce this pigment including the eye, the gastrointestinal tract, genitalia, sinuses, and meninges, in addition to the skin . The mosDuring the development of translation research related to MM, several disease models have been used to obtain fundamental discoveries in MM biology and these studies have resulted in the development of new therapeutic targets and the identification of several clinical markers. For example, several studies using animal models have revealed the efficacy of using anti-PD-1 and anti-CTLA-4 antibodies to inhibit tumor immunity ,12,13, lIndependently, to establish in vitro models that replicate several disease states, by using a 3D drop cell culture method, we recently succeeded in producing 3D spheroids of several non-cancerous cells including 3T3-L1 preadipocytes, human orbital fibroblasts (HOF) , human tTherefore, to study this issue, to elucidate the unidentified molecular mechanisms responsible for inducing the 3D spheroid architecture, we prepared 3D spheroids from five different MM cell lines  and dabrafenib and trametinib resistant A375 (A375DT) by a 3D drop cell culture method and the resulting spheroids were subjected to real time cellular metabolism analysis to evaluate the biological activities. In addition, among these five cell lines, we selected two distinct MM cells, one of which was horizontally circular shaped and the other non-horizontally circular shaped and subjected them to RNA sequence analysis to elucidate possible master regulatory genes responsible for these structural diversities.Five human MM cell lines including (1) A375 , (2) SK-mel-24 , (3) WM266-4 , (4) SM2-1  , and (5)A375DT (A375-dabrafenib/trametinib resistant) cell line was established from A375 cells by chronic treatment with gradually increasing concentrations of dabrafenib and trametinib, as described previously , and werOxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of the 2D cultured MM cell lines including SK-mel-24, SK-mel-23, MM418, A375, WM266-4, and SM2-1 were measured using a Seahorse XFe96 real-time metabolic analyzer  according to the manufacturer’s instructions  and a micro-monitoring camera equipped with a micro-squeezer  as described previously .® P330  and an Agilent 2100 Bioanalyzer , respectively. As RNA quality was suitable for RNA sequencing, and quantitative real time PCR, the samples with an RNA integrity number (RIN) > 8.5 were confirmed in advance. The total RNA was depleted of ribosomal RNA using NEBNext® Poly(A) mRNA Magnetic Isolation Module . The rRNA-depleted RNA was processed according to the manufacturer’s protocol to convert to cDNA using a TruSeq RNA Sample Preparation Kit  and final sequence-ready libraries with the NEBNext Ultra II RNA library prep kit . Then their quality and quantity were determined using an Agilent 2100 Bioanalyzer and KAPA Library Quantification Kit , respectively. Thereafter, they were subjected to NovaSeq 6000 and GenoLab M sequencing in PE150 mode. Sequence data were filtered by removal of the adapter sequence, ambiguous nucleotides, and low-quality sequences using FastQC (version 0.11.7) and Trimmomatic (version 0.38) software. Then, these clean reads were mapped to the reference genome sequence with a perfect match or one mismatch method via HISAT2 tools software [https://ftp.ensembl.org/pub/release-101/fasta/homo_sapiens/dna, accessed on 5 September 2022. The read counts for each respective gene and statistical analysis of the differentially expressed genes were calculated by featureCounts (version 1.6.3) and DESeq2 (version 1.24.0), respectively. Statistical significance was determined by the empirical analysis, and genes with fold-change ≥ 2.0 and FDR adjusted p < 0.05 and q < 0.2 were assigned as differentially expressed genes (DEG).Total RNA isolation was performed from 2D confluent cells ofWM266-4 and SK-mel-24 in a 150 mm dish as above (n = 3) using a RNeasy mini kit  according to the manufacturer’s instructions. RNA content and quality were measured using a NanoPhotometersoftware . The corhttps://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis, accessed on 5 September 2022) [A gene ontology (GO) enrichment analysis  as well er 2022)  were perer 2022) .For the knockdown of KRAS and/or SOX2 of SK-mel-24 cells, 2D cultured cells were incubated with Lipofectamine 3000  containing either 30 nM of (1) siRNA Universal Negative Control , (2) KRAS siRNA-1 , (3) KRAS siRNA-2 , (4) SOX2 siRNA-1 , (5) SOX2 siRNA-1  or (6) both KRAS siRNA-1 and SOX2 siRNA-2 for 48 h. according to the manufacturer’s protocol. After replacing the siRNA-Lipofectamine 3000 complexes media with normal growth medium to reduce toxicity, the cells were subjected to real time cellular metabolic analyses using a Seahorse XFe96 real-time metabolic analyzer , or a further 3D spheroid culture, as above.Total RNA extraction, reverse transcription, real-time PCR, and the quantification of the respective genes using specific primers  were desTo establish 3D human MM spheroids, human MM cell lines including WM266-4, SM2-1, A375, MM418, and SK-mel-24, were subjected to a 3D drop culture by which we recently succeeded in producing globe-shaped 3D spheroids of several non-cancerous cells including 3T3-L1 preadipocytes , human oTo elucidate functional aspects related to these five MM cell lines, as their cellular mitochondrial and glycolytic function, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were simultaneously monitored in real-time to estimate mitochondrial respiration and glycolytic ability by using a Seahorse XFe96 Bioanalyzer. As shown in To investigate the unidentified and underlying mechanisms responsible for inducing such a horizontal deformity in the 3D spheroid configuration among these human MM cell lines, the two most contrasting cell lines, MW266-4 and SK-mel-24, in which the deformity was minimal and maximal, respectively, were subjected to an RNA sequence analysis. As shown in the heat map A and MA To elucidate the possible functional roles of the above DEGs that were detected, a GO enrichment analysis and an ingenuity pathway analysis (IPA)  were employed. Among the categories of diseases and disorders, and molecular and cellular functions, the IPA analysis indicated that cancer and several cellular functions including organismal injury and abnormalities, endocrine system disorders, gastrointestinal diseases, and neurological diseases were most likely involved, based upon obtained DEGs . In addiTo elucidate which regulatory mechanisms are involved within the diversity between 3D architectures of both MM cell lines, a GO analysis and IPA up-stream and causal network analyses were performed. The results of the GO analysis based on up-regulated and down-regulated DEG suggest that several cellular mechanisms may be related, as shown in KRAS (up-regulated except 2D A375), SOX2 (down-regulated except 3D SM2-1), STAT3 , BRAF , FOS , MITF , (2) PCG1α (down-regulated), and (3) ECMs and TJ related molecule; COL1 (down-regulated except for up-regulated 2D SK-mel-24), COL6 (down-regulated except 3D A375), FN (down-regulated except for 3D SM-21 and up-regulated 2D MM418), aSMA (down-regulated except 3D MM418) and ZO1 (down-regulated except A375). Therefore, based upon these observations, we speculate that in addition to KRAS and SOX2, several additional oncogenic signaling related factors, namely, PCG1α, ECMs, and TJ related molecules may be related to the unidentified mechanisms for inducing differences in the horizontally deformed 3D MM spheroids. In addition, since these oncogenic signaling related factors that were evaluated as above are closely related to the pathogenesis of MM, such morphology-dependent alteration of their gene expression rationally suggests that anti-MM drugs may also affect 3D MM spheroid morphology.To investigate this further, mRNA expression analyses of several possible linked molecules including (1) oncogenic signaling related factors , (2) PCG1α, and (3) ECMs  and tight-junction (TJ) related molecule (ZO-1) were carried out among 2D and 3D cultured five MM cell lines . ComparePCG1α (3D), COL4 (3D), FN (2D), and αSMA (2D and 3D)  cascade regulating cellular proliferation, differentiation, and survival signaling . Based u and 3D) .Therefore, these collective data reported herein suggest that it is possible that the critical regulatory factors KRAS and SOX2, may significantly be influenced, not only by the 3D architectures of MM cell lines, but also by their physiological and pathological characteristics with several oncogenic signaling related factors as well as cell architecture supporting molecules.Although several biological aspects of 3D cultures of MM cell lines have been extensively characterized and are now used as in vitro models for studying the pathological and therapeutic significance of MM ,48, studAs possible mechanisms responsible for the diversity between the 2D and 3D cultures of 3T3-L1 preadipocytes, our recently reported transcriptome analysis using an IPA upstream analysis identified STAT3 as the master upstream gene as the regulator responsible for inducing the diverse biological properties between these culture systems . Among tHowever, as of writing, this conclusion remains speculative and the following study limitations would need to be investigated regarding these interesting and unidentified issues. These issues also include the relationship between horizontal and vertical deformity of the 3D MM spheroid and cellular physiology, the degree of malignancy, drug resistance, and others. Thus, to understand the biological correlations between 3D spheroid configurations and the tumor pathogenesis of MM in more detail, additional investigations with the objective of identifying additional unidentified factors related to KRAS and SOX2 will be our next projects.


One of the main problems in chemotherapy using platinum drugs as anticancer agents is the resistance phenomenon. Synthesizing and evaluating valid alternative compounds is challenging. This review focuses on the last two years of progress in the studies of platinum (II)- and platinum (IV)-based anticancer complexes. In particular, the research studies reported herein focus on the capability of some platinum-based anticancer agents to bypass resistance to chemotherapy, which is typical of well-known drugs such as cisplatin. Regarding platinum (II) complexes, this review deals with complexes in trans conformation; complexes containing bioactive ligands, as well as those that are differently charged, all experience a different reaction mechanism compared with cisplatin. Regarding platinum (IV) compounds, the focus was on complexes with biologically active ancillary ligands that exert a synergistic effect with platinum (II)-active complexes upon reduction, or those for which controllable activation can be realized thanks to intracellular stimuli. Cancer is one of the most lethal diseases, causing millions of deaths worldwide ,2. In paAdditional studies have led to the discovery of a second and third generation of platinum complexes. Respectively, these are carboplatin, which possesses higher solubility and stability than cisplatin, and oxaliplatin, which is water-soluble and exhibits a broader spectrum of activity than other complexes.Starting from cisplatin’s structure, some ligand modifications that could improve the complex’s biological properties have been introduced ,30.This review aims to go through all the progress of the last two years regarding the design of platinum complexes, both as Pt(II) and Pt(IV), used as anticancer agents in the treatment of cisplatin-resistant and orphan tumors .1) with heterocyclic thionate ligands has been synthesized  complexes. The advantage of this approach is that there are different structural and DNA-binding properties in comparison with cisplatin and its derivatives, which notably possesses a cis arrangement of ligands around the metal center. For this reason, a series of trans-Pt(II) complexes (complex thesized  31]..1) with 2, a dichloride Pt(II) complex counterpart in a cis conformation  and studied for their antiproliferative activities in comparison with cisplatin and complex ormation .1a showed statistically significant different activity compared with cisplatin, with significantly lower IC50 values, 4.31, 6.23, and 4.80 μM, compared with 9.71, 14.48, and 11.59 μM for cisplatin against A549, SKOV3, and MCF-7, respectively. Moreover, and most importantly, all complexes showed selectivity between tumorigenic and nontumorigenic cell lines. Interestingly, complex 2 showed lower antiproliferative activity  than others; complex 1b and, above all, 1a showed an ability to induce apoptosis in cancer cells with the highest potency, such that 1a has been used as the representative compound of the series in further studies. In addition, 1a can target the genomic content of MCF-7 cancerous cells and directly interact with DNA, as confirmed by comet assay. However, further studies are needed to identify the underlying mechanism of action of these platinum drugs within biological systems.All the new complexes showed cytotoxicity in vitro against the three cancer cell lines, but only complex 1a–e, trans-Pt(II) complexes with triphenylphosphine ligands have also been synthesized, and in particular, complexes 3 and 4 have been evaluated for their promising anticancer activities  with the same dialkylamino ligand preserved, in addition to the two chloride ligands. For all the performed studies, cisplatin was used as a reference and 3 and 4 were used as comparisons.Analogously to complexes tivities  32]. In. In1a–e,3′s results were the most effective due to the presence of N,N-dialkylamino side chains, which contributed to an increase in lipophilicity in the complex. This is an important aspect to evaluate, as it could be useful in overcoming one of the primary issues involved in cisplatin resistance, i.e., low cellular accumulation. The Pt content found in cells incubated with 3 was found to be higher than the reference drug, suggesting the ease 3 has in crossing the cell membrane.Complex 5, complex 3 has a higher cytotoxic effect: the IC50 values were 0.49 and 1.85 μM compared with 3.55 and 3.63 μM, seven and two times lower than complex 5 in sensitive and resistant ovarian carcinoma cells, A2780 and A2780cis, respectively. These results underline that the triphenylarsine group is more effective than the ancient triphenylphosphine group in eliciting cytotoxicity.Furthermore, in comparison with complex 3 showed the ability to covalently link with the target, but to a lesser extent in comparison with the reference drug, cisplatin. In addition, this cytotoxicity was related to the ability of complex 5 and complex 3 to interfere with the activity of topoisomerase II, an important enzyme involved in DNA metabolism and correct functionality. In particular, 3 behaved as a topoisomerase II poison. This detrimental effect on topoisomerase II, along with its ability to induce transmembrane depolarization in mitochondria, is primarily responsible for the cytotoxic effect of the complex, and it can promote cell death through the apoptotic pathway.In studying platinum complex interactions with salmon testis DNA, complex Another trans-conformation Pt(II) complex that can be used to overcome cisplatin resistance and its side effects is an inhibitor of the Hedgehog (Hh) pathway based on GANT61  33]. Th. Th33]. trans-[Pt(II)Cl2(dmso)L]-type complexes, 7 and 8, where L represents both GANT61 and 4-PCA, have also been synthesized  but without selectivity for the CSC-enriched cells and normal cells. It also showed positive activity against mammospheres, the multicellular, three-dimensional structures formed by CSCs: a significant reduction in their formation, size, and viability could be observed. Complex 7 induced apoptosis against two triple-negative breast cancer (TNBC) cell lines, MDA-MB-231, and BT549, with a potency that is, respectively, thirty and three times higher than complex 8 . A possible explanation for this activity is related to the presence of GANT61 as an N-donor ligand in complex 7; according to the abovementioned hydrolysis process  population, using salinomycin as the positive control and cisplatin and carboplatin as the Pt compound control. Complex  process , it releSince the aim is to improve the anticancer effects of conventional platinum-based drugs, another possible strategy is selectively introducing structural changes around the active platinum center by choosing either carrier ligands endowed with biological activity by themselves or substituting common chloride ligands.In this case, new platinum(II) complexes with 1-alkyl-1H-pyrazole ligands containing both iodide- and chloride-leaving groups have been synthesized  34]..34].9a and 9b, were more effective than the chloride analogs, 10a and 10b, and cisplatin against all cisplatin-resistant cell lines; while the chloride complexes had an effect comparable to cisplatin: 11 and 11.9 μM vs. 4 μM in the A2780 cell line; 21 and 19 μM vs. 22 μM in the A2780cisR cell line; 29 and 25 μM vs. 24 μM in the MDA-MB-231 cell line. These differences show that the mechanism of action of the new alkyl pyrazole complexes is different from that of cisplatin, thus potentially allowing the compounds to overcome the resistance mechanism. Another important aspect is that the new Pt(II) complexes are more effective in tumor cells with respect to healthy cells, thus confirming their higher selectivity toward tumor cells. Coherent with the antiproliferative effect, the intracellular Pt amount increases with lipophilicity: the most lipophilic complex causes the ethyl group to bond to the pyrazole and the iodide-leaving ligands. However, even if the amount of Pt from complexes 9a and b was about ninety-fold higher than cisplatin, their effectiveness would only be five-fold higher than that of cisplatin (4.2 and 5.0 μM vs. 24 μM in the MDA-MB-231 cell line). This means that a higher amount of Pt(II) alkyl pyrazole complex is necessary to obtain a certain level of biological activity compared with cisplatin.It was discovered that both iodide complexes, 9a,b and 10a,b. The experimental evidence shows that the contribution exerted by the unrepaired DNA lesions  on cytotoxicity was minimal compared with cisplatin. Conversely, it was found that the time-dependent cell response profiles (TCRPs) for complexes 9a,b and 10a,b, which can predict the mechanism of action in biologically active compounds, significantly differed from those typically expressed by DNA-damaging agents, such as cisplatin. Their profiles were instead superimposable on those displayed by nontubulin-targeting mitotic inhibitors toward Eg5. The mitotic tubulin Eg5 is an essential spindle motor protein, and it assembles and maintains the bipolar spindle during mitosis. When inhibited, a monoastral spindle forms, thus resulting in mitotic arrest. In the literature, it is reported that pyrazoles are important and potent inhibitors of Eg5, so analyzes have been performed to ascertain whether all the new Pt(II) complexes, 9a,b and 10a,b, cause a cell cycle arrest in the G2/M phase. This plausible molecular target also explains the selectivity for tumor cells exerted by these complexes, since healthy cells do not divide or only slowly proliferate tumor cells.These data suggest a new anticancer mechanism compared with cisplatin, whose main target is nuclear DNA. Binding studies using calf thymus DNA have revealed that both chloride and iodide complexes can covalently interact with the target, but although iodide binds DNA more efficiently than its chloride analog (as demonstrated by ICP-MS), the DNA platination occurs in both to a lesser extent than cisplatin. These results suggest that DNA might not be the main target of the new complexes, 11) and [Pt(MQ)(L)] (12), were synthesized, and their cytotoxicity was evaluated, in which tropolone was combined with 8-hydroxy-quinoline (Q) or 2-methyl-8-hydroxy-quinoline (MQ), two pyridinic scaffolds known for their cytotoxic activity in different types of tumor cell lines  complexes based on natural products, which have been recognized for their biological activity. Tropolone derivatives could be a significant example consistent with this idea . Tropololl lines .11 showed high cytotoxicity in these cell lines, especially T24 (IC50 = 3.6 μM), but also significant cytotoxic activity against the normal cell line, HL-7702; complex 12 showed selectivity in the tested cell lines, such as T24 and MGC80-3 , and lower cytotoxicity than cisplatin in the normal cell line. Overall, complex 11 showed an effect against T24, while complex 12 showed effects against both T24 and MGC80-3. The possible anticancer mechanism of both complexes, but especially complex 11, may be the production of high ROS levels through cell mitochondria, which can damage organelles and induce severe dysfunction in the cells’ machinery. In addition, complex 11 was proven to induce apoptosis by decreasing mitochondrial membrane potential, with a simultaneous increase in the Ca2+ level in the cells contributing to the onset of mitochondrial dysfunction, and, finally, by activating caspase-3 and caspase-9. Furthermore, complex 11 induced cell death in the G2 phase, downregulating the expression of Cdc25A, CDK1, cyclin A, and cyclin B and upregulating the expression of p27 and p21, proteins that interact with cyclin–CDK complexes within the nucleus and, hence, modify cell cycle progression. On the contrary, complex 12 induced cell arrest in the G1 phase and had similar effects to 11, but it lacked the influence of p27 expression. Complex 11 showed a significant in vivo anticancer effect, revealed by its inhibiting effects on tumor growth in T24 xenograft mice without any renal pathological changes or influence on renal function.The cytotoxicity was tested on different cancer cell lines: HeLa, T24, A549, NCI-H460, and MGC80-3, as well as on HL-7702 . Complex 13–18) were synthesized to overcome cisplatin resistance  complexes (sistance  36]..13–18) w13–18 was then evaluated using an MTT assay. It was discovered that the anticancer effect was improved when R1 was a methyl (14), phenyl (15), o-toluene (16), or tert-butyl (17), and the best activity occurred when both R1 and R2 were methyl groups. Furthermore, the different substitutions of R1 and R2 also influenced the accumulation of Pt agents in cancer cells, such that the anticancer activity could be modified by regulating the lipophilicity of the ligands.The antiproliferative activity of 50 values from 5.02 to 15.32 μM vs. cisplatin value of 36.58 μM). The higher concentration of Pt inside the cells correlates with higher anticancer activity.To confirm the ability of these complexes to overcome cisplatin resistance, they were analyzed in comparison with cisplatin. After 48 h of incubation, the antiproliferative activity of these Pt complexes is two- to seven-fold higher than the activity of cisplatin against cisplatin-resistant lung cancer (A549cisR) cells  complexes containing the triphenylphosphine moiety (widely used as a mitochondriothropic moiety), the dialkylamino group, and the bromide groups were synthesized  37]..37].19 and 20 in resistant cell lines, but, at the same time, complex 19 has proven to be more effective, underlying the beneficial effects of the dissymmetrical substitution pattern between these two brominated triphenylphosphine trans-platinum derivatives .It is worth noting that the substituents of the amino group are fundamental to the cytotoxicity of both 19; after 180 min, the uptake of cisplatin does not change, but the amount of complex 19 increases threefold. Indeed, while cisplatin probably undergoes an efflux effect, compound 19 shows a time-dependent accumulation in cells and a greater ability to cross the membrane. DNA interaction has been confirmed for both complexes due to the presence of the triphenylphosphine hydrophobic moiety and the trans geometry of bromide groups. However, this is not the only target that could explain the cytotoxic effect. Different biological assays support complex 19′s ability to depolarize more mitochondrial membranes than cisplatin under the same concentrations and experimental conditions, thus confirming that it interferes with the cellular redox state. Both complexes 19 and 20 can decrease the total thiols in both cisplatin-resistant and -sensitive cell lines, with cisplatin influencing the thiol concentration to a lower extent and only in the sensitive cell line. In addition, another mechanism of cytotoxicity is related to its influence on thioredoxin reductase (TrxR) activity. TrxR, along with glutathione reductase (GR), is a key enzyme in redox regulation and antioxidant responses inside cells. High levels of TrxR are one of the main characteristics of cisplatin-resistant tumor cells that need to be defeated. In cisplatin-resistant cells, complexes 19 and 20 can both affect the responsivity of TrxR, resulting in a lower amount than in untreated cells.While studying the mechanism of action of these two complexes, some differences with cisplatin have been found. First of all, from the cell uptake point of view, after 60 min of incubation, the amount of Pt is comparable with cisplatin and The purpose of synthesizing monofunctional cationic Pt(II) complexes is to produce substrates that benefit from binding to organic cation transporters (OTCs). This allows them to be used in the selective treatment of tumors where this transporter is overexpressed. Indeed, most of these transporters display high tissue specificity and subcellular or peculiar expressions, thus translating into specific tumor efficacy. To reach this goal, a series of cationic triamine platinum compounds have been synthesized. The general formula is [Pt(N-N′)N′Cl]X, where N–N′ is an aminomethyl–imidazole ligand, and N′ is an imidazole ring bearing the same alkyl group in the N1 position  38,39,4,439,40.24 is more effective compared with cisplatin, other ligands have also been studied, such as 8-aminoquinoline and its chiral 5,6,7,8-tetrahydro-derivatives, also called CAMPY  complexes. These differences are principally found in the accumulation of the complexes in different cellular phases, which results in a different effect on DNA: they seem to arrest cells into a G0/G1 phase to a greater extent; they more efficiently induce p53 mRNA; and α-tubulin and β-actin levels significantly reduce in response to new platinum(II) complexes. Furthermore, it should be highlighted that 28 (Pt-8AQ) has been further analyzed against six tumor cell lines. Three cell lines of human glioblastoma were present, i.e., U87-MG (expressing wild-type p53), U373-MG, and U138-MG (expressing endogenous mutant p53)  has recently been synthesized and evaluated for promising TNBC activity and theranostic properties (2(BrCat)) or the dianionic form of alizarine (H2(Aliz)) . Di. Di57]. hy cells .This review summarizes the recent advances in the synthesis of both platinum(II) and prodrugs platinum(IV) complexes for cisplatin-resistant cancer cell lines, whose therapeutic treatment still represents a great challenge. This can be correlated to the lack of selectivity toward the desired target and, therefore, the systemic side effects that often limit their final clinical approval. In this regard, despite continuous efforts in this direction, cisplatin remains the lead compound for so-called orphan tumors. The inability of researchers to bypass these problems can be linked to the incomplete unraveling of the mechanism of action of cisplatin, which is unknown in many ways. In recent years, ,59,60,61


BCR::ABL1 fusion in p190 isoform is a marker for worse prognosis, associated with treatment resistance and reduced overall survival. We aim to determine the effectiveness of targeting poly-ADP-ribose polymerase (PARP) in a model of BCR::ABL1 p190+ ALL using a small molecule inhibitor, AZD2461, and we hope that our findings may help improve molecular stratification and prognosis of ALL patients.Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and the presence of BCR::ABL1 fusion, is still a marker of worse prognosis for acute lymphoblastic leukemia (ALL), with resistance to tyrosine-kinase inhibitor therapy being a major obstacle in the clinical practice for this subset of patients. In this study, we investigated the effectiveness of targeting poly-ADP-ribose polymerase (PARP) in a model of BCR::ABL1 p190+ ALL, the most common isoform to afflict ALL patients, and demonstrated the use of experimental PARP inhibitor (PARPi), AZD2461, as a therapeutic option with cytotoxic capabilities similar to that of imatinib, the current gold standard in medical care. We characterized cytostatic profiles, induced cell death, and biomarker expression modulation utilizing cell models, also providing a comprehensive genome-wide analysis through an aCGH of the model used, and further validated PARP1 differential expression in samples of ALL p190+ patients from local healthcare institutions, as well as in larger cohorts of online and readily available datasets. Overall, we demonstrate the effectiveness of PARPi in the treatment of BCR::ABL1 p190+ ALL cell models and that PARP1 is differentially expressed in patient samples. We hope our findings help expand the characterization of molecular profiles in ALL settings and guide future investigations into novel biomarker detection and pharmacological choices in clinical practice.Detection of t, and consequent  BCR activator of RhoGEF and GTPase::ABL proto-oncogene 1 (BCR::ABL1) , acquired through Abcam® , was diluted in PBS 1× solution in a 1:1000 ratio, as well as the isotype control Recombinant Alexa Fluor® 647 Rabbit IgG monoclonal [EPR25A], being added to the samples and incubated for 30 min in the dark at room temperature. Samples were pelleted and resuspended in 400 µL of PBS 1× solution and the fluorescence intensity was analyzed in the flow cytometer BD FACSymphonyTM .Cells were fixed and permeabilized utilizing the kit eBioscience™ Foxp3/Transcription Factor Staining Buffer Set . Data were examined from the mean and standard deviation of three individual experiments utilizing either the Modfit LTTM (version 5.0) or the FlowJo (Version 10.8.1) software. To detect significant differences between the control and treated groups, the data were compared through analysis of variance (ANOVA) followed by Bonferroni’s post-test. Significant differences were considered with an interval of confidence of 95% (p < 0.05). GraphPad Prism 5.01 software  was used for data analysis and graph design.The emitted fluorescence was analyzed through ten thousand events detected through flow cytometry , following the instructions provided by the manufacturing company. Extracted RNA was quantified utilizing the equipment NanoDrop  and 20 ng was used for cDNA confection using the High-Capacity cDNA Reverse Transcriptase kit .Total RNA was extracted with TRIzol ReagentPARP1 (Reference: Hs00242302_m1), BCR::ABL1 p190 (e1a2) (Reference: Hs03024844_ft), and ACTB (Reference: Hs01060665_g1) genes were synthesized by the company ThermoFisherTM Scientific and real-time quantitative PCR (qPCR) reactions were carried out through the TaqMan® Gene expression assays . Amplified fragments were quantified in the equipment QuantStudio® 5 (Applied Biosystems®).Primers for the −ΔΔCT method, utilizing either the non-neoplastic cell line MRC5 or the non-treated controls as calibrators [p < 0.05). GraphPad Prism 5.01 software  was used for data analysis and graph design.Experiments were performed in triplicates and the relative expression levels were calculated through the 2ibrators ,17. Data® Genomic DNA Purification kit , and purity and integrity were assessed on the Agilent 2200 TapeStation  with D1000 ScreenTape (Agilent Technologies), according to the respective manufacturer’s protocol. Only samples with DNA Integrity Number (DIN) > 7 were used for downstream analyses.Cell line DNA was extracted using the Wizardhttps://visualization.ritchielab.org/phenograms/plot (accessed on 6 February 2023)) [Array-CGH (aCGH) experiments were performed on an Agilent microarray platform (Agilent Technologies) with a SurePrint G3 Cancer CGH + SNP Microarray 4 × 180 K slide (Agilent). Sample preparation, labelling, and microarray hybridization were performed according to the Agilent CGH Enzymatic Protocol version 7.5. Slides were scanned using the Agilent G2565CA scanner. Data were extracted with Feature Extraction software (v9.1 Agilent Technologies) and analyzed with Genomic Work Bench 11.0.1, Agilent Cytogenomics 5.0 and GeneSpring GX 14.5, as described elsewhere [y 2023)) .p  <  0.05 was considered as statistically significant. We used a Manhattan plot to visualize the enrichment results.Functional enrichment analysis of the CNAs was performed using the R package g:Profiler2  for geneBCR::ABL1 p190 through analysis of the buffy coat genetic material and had their PARP1 expression levels also quantified. All molecular assays went as previously described in Topic 2.5 utilizing RNA from the buffy coat of either peripheral blood or bone marrow. Immunophenotypic and karyotype data were acquired directly from each patient’s medical records.Blood samples were collected from adult patients either at the time of their diagnosis, with ALL or CML, or during medical treatment at the Hospital Geral de Fortaleza (HGF) and at the Ophir Loyola Hospital. This study was approved by the Ethics Committee of the Federal University of Ceará  and of the Ophir Loyola Hospital , informed written consent was obtained from the patient being brought into the study only after understanding and accepting the terms, and all methods were carried out in accordance with Helsinki guidelines and regulations. A total of 60 patient samples were deemed positive for the presence of PARP1 expression, analyses were made by dividing the cohorts into either ALL or CML patients, and data were analyzed through an average of the fold change expression of the patients in each cohort. Comparisons were made with blood samples from 10 healthy controls utilizing Student’s t-test for statistical analysis.Gene expression was quantified as described in Topic 2.5.1. To determine the significance of https://www.ncbi.nlm.nih.gov/gds (accessed on 6 February 2023)), a public repository of high-throughput gene expression data, to profile PARP1 expression in ALL. We downloaded and analyzed, as described elsewhere [We used the Gene Expression Omnibus (GEO) . T-tests or Wilcoxon tests were performed for analyses with two groups, and the significance level was set at PARP1 expression to the single-cell level, we used the Tumor Immune Single Cell Hub 2 (TISCH2) database [To extend our analysis of database . We seledatabase , which cdatabase , which idatabase  comprisidatabase ,28,29.PARP1 in leukemic cell lines relative to the non-neoplastic cell model reveals significant overexpression in both SUP-B15 (p < 0.0001) and Raji (p < 0.05), with incremental fifteen- and seven-fold changes in expression, respectively, while the PARP1 expression level in the cell line Namalwa was very similar and not statistically different from the control (PARP1 when compared with either of the neoplastic cell lines (p < 0.01), with Raji also presenting significantly higher expression when compared with Namalwa (p < 0.05).Comparative expression of  control . SUP-B151 nanomolar (nM).The use of doxorubicin, as expected, was met with significantly lower values of IC50 than the molecular targeted therapies due to the non-specificity of its action over the viability of rapidly dividing cells , presentBCR::ABL1 translocation, the inhibitory activity of imatinib over SUP-B15 is not a surprise and has already been described in the literature [In the SUP-B15 cell line, the IC50 values of AZD2461 and imatinib were low and extremely similar to one another, demonstrating almost equivalent inhibition potential. Due to harboring terature ; howeverIn the cell lines Raji and Namalwa, used as comparative controls of other B-cell neoplasms with different PARP1 expression levels, the 72-h IC50 of AZD2461 and imatinib showed great disparity. In both cell lines, imatinib presented some cytotoxic activity, probably due to its non-specific activity over other tyrosine kinase pathways that are essential in the maintenance of proliferative capabilities and in the survival of neoplastic clones . HoweverDue to the non-responsiveness of Raji and Namalwa to the proposed treatment, cytotoxic assays of 48 and 24 h of treatment were only carried out with the SUP-B15 cell line, as well as all the subsequent phenotypic and molecular characterization assays. The obtained results are described in t-test, demonstrating the equivalence of both drugs in treating this ALL p190+ model in vitro.Taking into consideration the three treatment intervals, the inhibitory values of AZD2461 and imatinib in incubation with SUP-B15 were not significantly different when analyzed through Student’s p < 0.0001).Analysis of cell cycle arrest induced by treatment strategies allows for the identification of a drug’s potential cytostatic properties, inhibiting neoplastic clones proliferation ,36,37. FIn our non-treated control experiment, most events of the SUP-B15 cell line were detected on the G0/G1 phase—47.37%—with a great amount also detected on S phase—43.98%—and only a minority of cells undergoing mitosis on phase G2/M—8.66%. This low presence of SUP-B15 cells in the mitotic phase goes along with what is already described in the literature and is representative of the extensive duplication time of SUP-B15 of approximately 46 h . Furtherp < 0.0001), with 72,6% and 75,12% of events in G0/G1 to the treatments, respectively, and no statistically significant difference was detected in the arrest induced between both treatments. In both treatments, the arrest was induced with the detriment of S phase cells, being significantly decreased in comparison to the control (p < 0.0001), with 19.3% of events in the S phase for the AZD2461 treated experiment and 14.9% of events in S phase for the imatinib treated experiment, once again there not being detected a statistically significant difference between S phase events of both treatments. Finally, in the analysis of the G2/M phase, no difference was detected comparing either treatment with the control, with 8.36% and 9.98% of cells in the G2/M phase for AZD2461 and imatinib, respectively.In the experiments of AZD2461 or imatinib treatment, a significant arrest in phase G0/G1 was observed in comparison to the non-treated control  fluorochrome was used to identify exposed phosphatidylserine, a marker of early apoptosis, on the outer membrane of cells after the treatment incubation period ,41. Addip < 0.0001).The use of imatinib in sub-inhibitory concentrations of 1.5 µM did not have any effect of apoptosis induction after 24 h of incubation, not being statistically different from the non-treated control experiment. Meanwhile, the use of AZD2461 in the same concentration was able to induce a considerable increase in the frequency of both early apoptosis and late apoptosis/necrosis populations in comparison to control and to cells treated with imatinib  or imatinib (p < 0.0001) significantly increase BCR::ABL1 p190 expression levels in comparison to the non-treated SUP-B15 control group, 3- and 4.5-fold, respectively (p < 0.05), although the same tendency for BCR::ABL1 p190 mRNA increase was detected. While BCR::ABL1 overexpression predicts TKI-resistant phenotypes [BCR::ABL1 upregulation has been previously reported in CML cell models and is associated with neoplastic attempts to evade cell death [BCR::ABL1 upregulation as a compensatory mechanism to Ph+ ALL as well. The finding that AZD2461 also upregulates BCR::ABL1 p190 expression levels reveals a similar pattern of cell death evasion for both treatments.qPCR analysis reveals that both treatments with AZD2461 , while imatinib-treated populations had no statistical difference to the control group. A tendency for lower levels of PARP1 may be seen in the imatinib-treated group, however, as evidenced by an increase in statistical significance when comparing both treated groups to each other (p < 0.01).To prove if AZD2461 or imatinib treatments induced post-transcriptional modifications on PARP1 levels, cells were stained with anti-PARP1 antibodies and analyzed by flow cytometry for mean fluorescence intensity (MFI) quantification . CompariA clear trail may be seen in the AZD2461-treated graph, revealing a small percentage of events with depletion of PARP1 levels, and taken together results from the expression and PARP1 intracellular levels which seem contrasting. However, our flow cytometry analysis indicates that AZD2461 acts directly over PARP1 protein, inducing degradation and consequent loss of catalytic function, much sooner than it may act over gene expression.PAX5 at 9p13.2 (loss) [CDKN2A/B at 9p21.3 (loss) [MYC at 8q24.21 [PARP1 gene, localized in chromosome 1q42.12, supporting its high mRNA expression observed in the real-time PCR data  in the SUP-B15 cell line. Interestingly, this cell line harbored 28 CNAs (4 gains and 24 losses) with sizes ranging from 0.11 Mb to 86 Mb . Large C2 (loss) ,49,50, C 8q24.21   count, immunophenotype, karyotype, and qPCR cycle threshold (Ct), are reported in the Attempting to gather evidence of PARP1 relevance in the landscape of PARP1 may be seen, as evidenced by the significant twofold increase compared with healthy donors , T-cell ALL, and samples from the bone marrow of healthy donors ), which correspond to the amino acids R213Q and R248Q of the TP53 protein in Raji and Namalwa, respectively. In a study by Zhang et al. [Further exploring oncogenic pathways which are common to both cell lines and may help to overcome PARPi cytotoxicity, we highlight the presence of mutations in g et al. , R213Q ig et al. , diminisg et al. . As suchg et al. , and theg et al. , the samAnother characteristic shared among both cell lines of BL is the infection of Epstein-Barr virus (EBV), highly associated with the leukemogenesis of B-lymphocyte malignancies, being a major prediction factor of bad prognosis in clinical practice ,62. MorgBCR::ABL1 that drives Ph+ tumors such as SUP-B15 is described as able to induce leukemia onset and development by itself [BCR::ABL1 expression, a condition denominated “oncogene addiction” [BCR::ABL1 presence [When compared with the other two analyzed cell lines, SUP-B15 does significantly overexpress PARP1 and was extremely responsive to treatment with PARPi in levels comparable to those of the gold standard Imatinib . The tray itself , and Ph+diction” . In thispresence , the inhBCR::ABL1 p190+ B-cell ALL are determinant factors to the sensitivity to PARPis and not the inherent physiology of B-lymphocytes per se.Although AZD2461 does not have any tolerability tests in patients with published results, its use in vitro does not show cytotoxicity over the viability of B-lymphocytes, even in concentrations as high as 40 µM . TherefoOur cell cycle analysis reveals important cytostatic characteristics of both AZD2461 and imatinib, with similar profiles of significant arrest in G0/G1 in detriment of cells in phase S . In the Furthermore, the higher frequency of death marker presentation in assays treated with AZD2461 may reflect its activity over the previously mentioned tumoral subpopulations detected in the cell cycle analysis. Comparing the results from the cell cycle arrest and early death experiments, it may be interpreted that the cells marked precociously with annexin-V are representative of the same subpopulation that drastically diminishes in frequency in the cell cycle arrest analysis, indicating that AZD2461’s ability to induce early death does not encompass the whole of the SUP-B15 cell line, but rather its subpopulations. From the obtained results, characterization of the subpopulations which form the cell line through single-cell transcriptomic analysis or differential marking of membrane antigens is of extreme importance for proper stratification of risk groups inside this leukemic model and for the prediction of PARPi effectiveness.PARP1 expression in the SUP-B15 cell line is corroborated by the findings of our aCGH array analysis, revealing amplification of the long arm of chromosome 1, where PARP1 is localized (PARP1 seen in this population through single-cell transcriptomic analysis (PARP1. Furthermore, studies have demonstrated that the therapeutic association of PARPi with immune system pathway inhibitors is indeed promising in several types of tumors [The observation of high ocalized . Additioocalized . The PARanalysis A and thef tumors ,76,77,78MYC proto-oncogene (MYC expression and influence was not investigated in our study, it has been previously reported as a mediator of PARP1 activity, inducing increased activity of nonhomologous end-joining pathways [MYC-driven B-cell lymphoma, Galindo-Campos et al. [MYC mutational status and expression profile may also be a cofactor underlying the sensitivity of our model of p190+ ALL to PARP inhibition and future investigations are needed to fully elucidate this hypothesis.Also, of interest is the observed amplification of the long arm of chromosome 8, encompassing oncogene . While Mpathways ,80. In mpathways  demonstrs et al.  report dPARP1 in p190+ ALL patient samples is attested when looking both into our molecular profile analysis and into online patient sample datasets (PARP1 in p190+ CML, however, indicate that the presence of the p190 isoform alone is not enough to dictate high PARP1 expression, although the use of PARPis has already been demonstrated to have cytotoxic effect over BCR::ABL1 positive CML in vivo, even though PARP1 levels were not quantified [PARP1 expression and patient’s clinical features such as age, WBC, blast percentage, immunophenotype, and karyotype.Finally, the validation of the differential expression of datasets , corroboantified . AltogetBCR::ABL1 p190+ translocation were used as predictors of PARPi sensitivity in cohorts of B-cell neoplastic cell lines, with the observed similar cytotoxic profiles of PARPi, AZD2461, and gold standard treatment, imatinib, against a BCR::ABL1 p190+ ALL cell model fomenting the idea of utilizing PARP as a potential therapeutic target in this neoplastic model. Still, further in vivo studies and more extensive characterization of other BCR::ABL1 p190+ models are necessary to validate our hypothesis. Overall, we hope our findings help expand the characterization of molecular profiles in ALL settings and guide future studies into novel biomarker detection and pharmacological choices in clinical practice.In this exploratory study, high PARP1 expression and presence of


Background: Triple-negative breast cancer (TNBC) is an aggressive subtype with the most unfavorable clinical outcomes, in part due to tumor heterogeneity, treatment resistance, and tumor relapse. The TNBC subtypes  are biologically and clinically distinct entities that respond differently to local and systemic therapies. Therefore, we need to have a better understanding of cancer stemness relating to drug-resistant populations in the TNBC subtypes.Methods: Breast cancer stem cell (BCSC) distribution was investigated using an integrated flow cytometry approach with the ALDEFLUOR™ assay (ALDH) and CD24/CD44 antibodies. In total, 27 commercially available cell lines derived from normal and malignant mammary tissue were characterized into differentiated tumor cells and/or BCSC subpopulations .Results: BCSCs were not only enriched in estrogen receptor (ER) negative  and TNBC cell lines , but certain BCSC subpopulations  were also significantly more common in the M  and BL2  TNBC subtypes. In contrast, ALDH status alone was not indicative of ER status or BC subtype.Conclusion: Taken together, these findings demonstrate the enrichment of potentially treatment-resistant BCSC subpopulations in the M and BL2 triple-negative breast cancer subtypes. Triple-negative breast cancer (TNBC) is a heterogeneous disease comprised of four distinct molecular subtypes  with differing biology , therape+CD24−/low phenotype and high aldehyde dehydrogenase (ALDH+) activity, i.e., ALDH−CD44+CD24−/low enriched mesenchymal-like BCSCs , ALDH+non-CD44+CD24−/low enriched epithelial-like BCSCs , and highly purified ALDH+CD44+CD24−/low BCSCs with both mesenchymal and epithelial characteristics are considered to be the most tumorigenic . The cell lines were classified into the breast cancer molecular subtypes  as previously described (Twenty-four human breast cancer cell lines and a non-cancer cell line (MCF-10A) were purchased from the American Type Culture Collection  or Deutsche Sammlung von Mikroorganismen und Zellkulturen . Göran Landberg and Julie Grantham (University of Gothenburg) kindly provided the MDA-MB-468 and MCF-7 cells, respectively. The cells were maintained in a humidified atmosphere containing 5% COescribed . Cell liescribed . Authent-non-CD44+CD24−/low), highly purified BCSCs (ALDH+CD44+CD24−/low), enriched epithelial-like BCSCs (ALDH+non-CD44+CD24−/low), and enriched mesenchymal-like BCSCs (ALDH−CD44+CD24−/low) according to the CD44+CD24−/low phenotype and ALDHbr activity using the ALDEFLUOR™ Kit , PE-conjugated mouse anti-human CD24 antibodies , and APC-conjugated mouse anti-human CD44 antibodies . In brief, dry ALDEFLUOR™ reagent was suspended in DMSO, 2N HCl, and ALDEFLUOR™ Assay Buffer at room temperature. The fluorescent-activated ALDEFLUOR™ reagent was then aliquoted and stored at −20°C. Five cell lines  were used to determine the optimal sample concentration  and ALDEFLUOR™ incubation time  at 37°C. The optimal sample concentration was determined by identifying the sample concentration with the largest difference in mean fluorescence intensity (MFI) between ALDHbr (ALDEFLUOR™) and ALDHlow  cells. The optimal sample concentration was 1 × 105 cells/mL for all five cell lines, but the optimal incubation time was cell line-specific . For the remaining 22 cell lines, 1 × 105 cells/mL and 40 min incubation time were used.Flow cytometry was used to categorize each cell line into differentiated tumor cells  for 10 min at room temperature in the dark. Each sample was resuspended in 300 µL ALDEFLUOR™ Assay Buffer and stored on ice in the dark until analysis. After filtering, a minimum of 10,000 events were analyzed using a BD Accuri™ C6 Personal Flow Cytometer  and FlowJo™ v10.8.1 Software (BD Life Sciences). A minimum of two biological and technical replicates were performed on selected cell lines. The gating strategy is outlined in For each cell line, 2 × 10p-value cutoff in R/Bioconductor version 4.0.4. Values are presented as the mean and standard deviation (SD). The Shapiro-Wilks normality test was performed to determine whether the parametric T-test  or non-parametric Wilcoxon test  should be used. Plots were constructed using the moonBook (version 0.3.1)  , ggplot2n 0.3.1) , and ggpn 0.3.1)  packages-non-CD44+CD24-/low) were primarily found in Luminal A and HER2amp cells (−CD44+CD24−/low) were predominantly identified in the BL2 and M subtypes, while enriched epithelial-like BCSCs (ALDH+non-CD44+CD24−/low) were found in 16/27 (59.3%) cell lines. Although rare, highly purified BCSCs (ALDH+CD44+CD24−/low) were only identified in one HER2amp cell line (JIMT-1) and TNBC cell lines, particularly HCC1806 and MDA-MB-436 cells (both BL2 subtype). These findings demonstrate the existence of distinct BCSC subpopulations based on TNBC subtype.In total, 16/27 human breast cell lines included in the study were TNBC (59.3%), of which 23.5% were characterized as BL1, 25.0% as BL2, 12.5% as LAR, 31.2% as M, and 6.2% as TNBC unspecified . BCSC pomp cells . In TNBCp > 0.05; versus 0.8% ± 1.2 in BL2 and 1.0% ± 2.2 in M). Differentiated tumor cells were significantly more common in ER+ (93.1% ± 11.8 versus 50.5% ± 37.6 in ER-), Luminal A (97.9% ± 2.4 versus 48.7% ± 38.0 in TNBC), and BL1 TNBC . In contrast, enriched mesenchymal-like BCSCs were predominantly found in ER- (40.2% ± 40.8 versus 0% ± 0 in ER+), TNBC , as well as BL2 (77.4% ± 21.4 versus 6.2% ± 11.5 in BL1) and M TNBC cells (64.0% ± 42.7 versus 6.2% ± 11.5 in BL1). Although 2/4 BL2 cell lines showed the highest distribution of highly purified BCSCs (9.3% in HCC1806 and 8.8% in MDA-MB-436 cells), significantly more highly purified BCSCs were generally found in ER- .Further examination of the distribution of differentiated tumor cells and BCSC subpopulations in the 27 cell lines revealed no significant differences in enriched epithelial-like BCSCs based on estrogen receptor (ER) status or BC subtype (br cells (ALDH+ and ALDH-) and the CD44+/CD24−/low phenotype (CD44+CD24−/low and non-CD44+CD24−/low) were then analyzed separately, thereby demonstrating that ALDH-negativity was prevalent in the majority of cell lines , TNBC status , BL2 (81.9% ± 23.2 versus 6.6% ± 11.9 in BL1) and M TNBC cells  were primarily found in Luminal A and HER2amp cells. Intriguingly, mesenchymal-like BCSCs were predominantly found in cell lines derived from ER- breast cancer and the M and BL2 TNBC subtypes, while epithelial-like BCSCs were detected in the BL1 and LAR TNBC subtypes. Used as separate BCSC biomarkers, the CD44+/CD24−/low phenotype was also more indicative of ER, BC and TNBC status than ALDH activity.Breast cancer stem cells have previously been shown to be enriched in estrogen receptor-negative and triple-negative breast cancer . This iset al. only studied two TNBC patient-derived xenografts without consideration of their TNBC subtype. Other studies either used one BCSC marker  or both in two separate experiments  without including the ALDEFLUOR™ assay.To our knowledge, only one other study has investigated BCSC distribution using this integrated approach in a single experiment . Howevereriments . Integraet al. recently illustrated that the BL2 and M subtypes may be derived from myoepithelial/basal cells, though the origin of the M subtype points to de-differentiated BL1 cells  and 100% ALDH-positivity for BT-474 (36% in the current study) by Zhou et al. . BCSC subpopulations were more prevalent in cell lines derived from ER-negative breast cancer, as well as the M and BL2 TNBC subtypes. However, further evaluation of BCSC distribution is warranted in TNBC patient biopsies (classified by the TNBCtype-4 molecular subtypes) in relation to treatment response and clinical outcome.


Even in the novel immunotherapy era, Merkel cell carcinoma (MCC) remains challenging in its treatment. Apart from Merkel cell polyomavirus (MCPyV) associated MCC, this cancer is linked in about 20% of cases to ultraviolet-induced mutational burden frequently causing aberrations in Notch and PI3K/AKT/mTOR signalling pathways. The recently developed agent GP-2250 is capable to inhibit growth of cells of different cancers, including pancreatic neuroendocrine tumors. The objective of the present study was to investigate the effects of GP-2250 on MCPyV-negative MCC cells.Methods We employed three cell lines  which were exposed to different GP-2250doses. GP-2250’s effects on cell viability, proliferation, and migration were evaluated by means of MTT, BrdU, and scratch assays, respectively. Flow cytometry was performed for the evaluation of apoptosis and necrosis. Western blotting was implemented for the determination of AKT, mTOR, STAT3, and Notch1 protein expression.Cell viability, proliferation, and migration decreased with increasing GP-2250 doses. Flow cytometry revealed a dose response to GP-2250 in all three MCC cell lines. While the viable fraction decreased, the share of necrotic and in a smaller amount the apoptotic cells increased. Regarding Notch1, AKT, mTOR, and STAT3 expression a comparatively time- and dose-dependent decrease of protein expression in the MCC13 and MCC26 cell lines was observed. By contrast, Notch1, AKT, mTOR, and STAT3 expression in MCC14.2 was scarcely altered or even increased by the three dosages of GP-2250 applied.The present study indicates GP-2250 having anti-neoplastic effects in MCPyV-negative tumor cells in regard to viability, proliferation, and migration. Moreover, the substance is capable of downregulating protein expression of aberrant tumorigenic pathways in MCPyV-negative MCC cells. Merkel cell carcinoma (MCC) represents a rare skin cancer with epithelial as well as neuroendocrine differentiation. In approximately 80% of cases, MCPyV is clonally integrated into the tumor whereas in the remaining 20% the tumors are associated with high rates of ultraviolet (UV)-induced mutations . Buchholz et al. , MCC14.2 (CBA-1340), MCC26 (CBA1341)]. On the basis of a material transfer agreement, the cell lines were provided to J.C.B. by J.H. Leonard . All cell lines have been maintained in RPMI-1640 supplemented with 10% Fetal Bovine Serum (FBS), penicillin/ streptomycin each 100 U/ml and 2 mM L-Glutamine. The culture media for MCC 14.2 and MCC 26 was additionally supplemented with 25 mM HEPES . The cells were maintained as monolayer at 37 °C with 5% COWe used powdered GP-2250, which was provided by Geistlich Pharma AG . The substance, dissolved in double-distilled water, was freshly prepared every other week and stored at room temperature by. It was set to a physiological pH, sterile filtered, and stored protected from light.The MTT assay was carried out on all cell lines in order to assess colometrically the anti-neoplastic effects of GP-2250. The MCC cells were individually seeded to obtain a sub-confluent monolayer in a 96-well plate format and were incubated for 24 h prior treatment. To examine its dose–response relationship, the MCC cells were exposed to concentrations ranging from 50 to 4500 µmol/l which depended on the cell line and ddH2O as control for 24 h. Following stated exposition times, 10 µl MTT -2,5-diphenyltetra-zoliumbromid) reagent (5 mg/ml) was added and incubated for 2 h before violet Formazan crystals were dissolved in 100 µl DMSO (Dimethyl sulfoxide). The cell viability was determined by means of a microplate absorbance reader  by determining the optical density at 560 nm (reference wavelength 720 nm). Using three independent experiments with consecutive passages, the MTT assay was performed with eight replicates.. Using three independent experiments with consecutive passages, BrdU assay was performed with eight replicates.The MCC cells were individually seeded to obtain a sub-confluent monolayer in a 96-well plate format and were incubated for 24 h prior treatment. To determine the dose–response of GP-2250 with respect to the anti-proliferative activity of the substance, the cells were exposed to concentrations of GP-2250 ranging from 50 to 3500 µmol/l. The exposure depended on the MCC cell line and ddH2O as control for 6 h and submitted to BrdU proliferation assay (5-bromo-2-deoxyuridine)-ELISA  according to the manufacturer’s instructions. The 6-h incubation time was appropriate for the BrdU assay as indicated by our previous investigations. The amount of synthesized DNA was measured by means of a microplate absorbance reader  at 450 nm with a reference wavelength of 550 nmTo form a confluent monolayer, all three MCC cell lines were plated into 60 mm dishes and incubated for 24 h. The needed number of cells for a confluent monolayer was dependent on the MCC cell type. All cell lines were exposed to GP-2250 concentrations ranging from 250 up to 4000 µmol/l. After introducing a scratch in the cell monolayer, mimicking a wound, the area was examined by phase-contrast-microscopy. Images of the scratch assay were captured at start of the exposures and at intervals  during the migration of cells closing the scratch, to semi-quantify the migration rates.Again, the MCC cells were individually seeded in order to obtain a sub-confluent monolayer in a 6-well plate format and were incubated for 24 h. Different individual concentrations of GP-2250 (from 100 up to 4000 μmol/l) and ddH2O (control) were utilized for 24 h before the cells were assessed by flow cytometry analysis. The latter was carried out in 4 to 6 independent measurements including 2 to 4 consecutive passages. The MCC sells were resuspended in a 200 μl binding buffer . Thereafter, the cell suspension was treated with 5 to 10 μl Annexin V-FITC  over 15 min at room temperature in the dark. Then, 10 μl of propidium iodide (PI)  were added. Subsequently, the cells were assessed by flow cytometry  for Annexin V-FITC and PI binding. The dot plots and histograms were evaluated by means of the CellQuest Pro software .Protein isolation was performed by RIPA (Radio immunoprecipitation assay) lysis . Subsequently a BCA (Bicinchoninic acid) assay  was used for protein quantification. After loading equal amounts of protein per lane (30 μg protein), 7 to 20% Protean-TGX (Tris–Glycine eXtended) gels , were electrophoresed at 250 V for 30 to 45 min and transferred on to a TransBlot Turbo PVDF (Polyvinylidene fluoride) membrane  using a TransBlot Turbo system . In line with the manufacturer’s antibody specification protocol, the membranes were blocked in EveryBlock Blocking buffer  over 5 min and incubated overnight at 4 °C with primary antibody  at 1:1000 dilution. Thereafter, the membranes were washed using PBST  and incubated with an Anti-rabbit IgG HRP-linked AB 7074; . Band detection was carried out using ChemiDoc MP imaging system . Comparative quantification of Western blot results was performed using the BIO RAD Image lab software, Version 6.1, .t test was employed for pairwise comparisons. p values ≤ 0.05 were considered statistically significant and are shown in the figures as follows: ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. Statistics were performed by means of Graph Pad Prism 9.1.0 .The data of the MTT assay , BrDU assay , scratch assay , and flow cytometry analysis  are given in mean ± SD. We used ANOVA including the Tukey’s post-hoc test for normally distributed data. Where appropriate, the Figure Correspondingly, all three MCC cells demonstrated a dose response to GP-2250 during BrDU to different range (Fig. Concerning flow cytometric measurements, all three MCC cell lines showed a dose response to GP-2250, its range varying between cell lines Fig. . Flow cyAs displayed in Fig. Western blotting was performed for protein expression analysis of AKT, mTOR, STAT3, and Notch1 in all three MCC cell lines. Cells were exposed to GP-2250 applied in three individual concentrations, having proven effective in previous experiments. Regarding AKT expression, there was a moderate time- and dose-dependend decrease of protein expression in MCC13 and MCC26 (Fig. As shown in Fig. Figure RB1 and TP53 mutations, aberrations occur also frequently in Notch genes as well as in the PI3K/AKT/mTOR signaling pathway which we have addressed in the present study (Dobson et al. The mutational tumor burden of MCC differs significantly between MCPyV-negative MCC and MCPyV-positive MCC. Virus-negative MCC has a very high overall mutational rate when compared to virus-positive MCC (Stachyra et al. Notch, thus Notch1 mutations are observed in up to 90% of MCC cases (Goh et al. AKT1gain-of-function mutants have previously been observed. There is are interactions between the AKT/mTOR signaling and several other pathways, such as the JAK‐STAT signaling pathway. Consequently, the activation of JAK2 results in activation of STAT transcription factors (Dobson et al. Notch1 signaling plays a significant role in differentiation, proliferation, cell fate, and programmed cell death. By contrast, signaling of Notch2 takes part both in cell fate determination in the embryo and regulation of the immune system. Both pathways have been shown to be affected in MCC. About ¾ of virus-negative MCC show alterations in The exact mode of action of GP-2250 remains yet to be fully established. However, GP-2250 is capable to deplete metabolic energy through inhibition of the enzyme GAPDH which is rate limiting for aerobic glycolysis (Braumann et al. TP53 mutation, whereas MCC13 originates from a cervical lymph node metastasis with TP53 mutations and MCC26 is presumably derived from a cutaneous primary tumor harboring HRAS and RB1 mutations (https://www.cellosaurus.org, access: January Indeed, limitations of the present study include the divergent findings of western blotting between MCC14.2 and the other two cell lines. This discrepancy can only be in parts explained by differences with respect to underlying tumor characteristics. The cell line MCC14.2 originates from an iliac lymph node metastasis harboring a Our preliminary data indicate that GP-2250 has anti-neoplastic effects in virus-negative MCC cells regarding tumor cell viability, proliferation, and migration. Moreover, GP-2250 is capable to downregulate protein expression of aberrant tumorigenic pathways in virus-negative MCC cell lines.


Natural polymer-based porous scaffolds have been investigated to serve as three-dimensional (3D) tumor models for drug screening owing to their structural properties with better resemblance to human tumor microenvironments than two-dimensional (2D) cell cultures. In this study, a 3D chitosan–hyaluronic acid (CHA) composite porous scaffold with tunable pore size  was produced by freeze-drying and fabricated into a 96-array platform for high-throughput screening (HTS) of cancer therapeutics. We adopted a self-designed rapid dispensing system to handle the highly viscous CHA polymer mixture and achieved a fast and cost-effective large-batch production of the 3D HTS platform. In addition, the adjustable pore size of the scaffold can accommodate cancer cells from different sources to better mimic the in vivo malignancy. Three human glioblastoma multiforme (GBM) cell lines were tested on the scaffolds to reveal the influence of pore size on cell growth kinetics, tumor spheroid morphology, gene expression and dose-dependent drug response. Our results showed that the three GBM cell lines showed different trends of drug resistance on CHA scaffolds of varying pore size, which reflects the intertumoral heterogeneity across patients in clinical practice. Our results also demonstrated the necessity to have a tunable 3D porous scaffold for adapting the heterogeneous tumor to generate the optimal HTS outcomes. It was also found that CHA scaffolds can produce a uniform cellular response (CV < 0.15) and a wide drug screening window (Z′ > 0.5) on par with commercialized tissue culture plates, and therefore, can serve as a qualified HTS platform. This CHA scaffold-based HTS platform may provide an improved alternative to traditional 2D-cell-based HTS for future cancer study and novel drug discovery. Adult glioblastoma multiforme (GBM) is one of the most deadly and recalcitrant cancers in the U.S. . For decCompared to cancer cells cultured with 2D monolayers, cancer cells cultured in 3D matrix are able to form self-assembled aggregates, aptly termed “tumor spheroids”, and render cell behaviors similar to those in in vivo tumor tissues . They haNatural polymers have been widely used in tissue engineering applications thanks to their excellent biocompatibility. Natural polymers, such as chitosan and hyaluronic acid (HA), contain similar chemical structures to glycosaminoglycan, a major extracellular matrix (ECM) constituent in the human body. These polymers are extensively studied and prove to be suitable for constructing 3D scaffolds for in vitro cell cultures as they provide a microenvironment that can closely mimic the human tissue ECM . CompareHowever, there are obstacles still holding the translation of such scaffold-based HTS platforms back, from bench-top research to human clinical trials. First, the polymer solution of chitosan and HA is usually highly viscous and difficult to handle, hampering the large-batch production of CHA scaffolds . Hence, In this study, we applied a self-designed rapid dispensing system to manage the viscous polymer solution of chitosan and HA and establish a fast and cost-efficient approach to manufacture the HTS platform. We prepared the CHA scaffolds using the freeze-drying technique, which is suitable for processing heat-sensitive natural polymers and capable of controlling the porous structure of the scaffold by adjusting freeze-drying parameters . Recent All chemicals were purchased from Merck , unless otherwise specified.w/w chitosan  and 1% w/w hyaluronic acid  were fully dissolved in 1% w/w acetic acid aqueous solution, respectively. Two solutions were then combined and mixed using a Thinky mixer  at 2000 rpm for three minutes, and further mixed in a blender for ten minutes to ensure a homogeneous polymer mixture. The polymer mixture was then centrifuged at 2000 rpm for 30 min to remove air bubbles and cast into 96-well tissue culture plates using a self-designed computer-aided automated scaffold dispenser [v/v ammonium hydroxide/methanol solution for 30 min under vacuum, rinsed intensively with DI water and soaked in PBS for 24 h to remove residual base. The scaffolds were then sterilized using 70% ethanol for 24 h and then washed with sterilized PBS three times and incubated in 37 °C for another 24 h prior to cell seeding.A total of 8% of CHA scaffolds were prepared similarly to a previously described method . A totalispenser . The disScaffold imaging and pore size characterization: Scaffolds were sectioned into 400 µm thin slices using a Compresstome VF-300 Vibrating Micromtome . Samples for SEM were sputter-coated with gold/platinum before imaging. SEM images were captured under 100× and 300× magnification using a JSM-6010 Plus scanning electron microscope . Samples for optical imaging were hydrated with PBS for 7 days before imaging. Optical images were captured using a MU-1000 optical microscope . The scaffold pore size was characterized by measuring the individual pore diameter using the ImageJ software program. At least 50 pores were measured for each scaffold.iV) and weight (iW) were first recorded. The scaffold was then immersed in isopropanol (with known density iρ) under vacuum until the scaffold stopped bubbling and sank to the bottom. The impregnated scaffold was carefully wiped off of excessive isopropanol and weighed again to obtain the final weight (fW). The change in the volume of the impregnated scaffold was deemed to be negligible as isopropanol is a nonsolvent. The open porosity was defined as the ratio of the volume of the solvent within the scaffold pores to the volume of the dry scaffold, as shown in Equation (1).Scaffold open porosity: The scaffold open porosity was measured using the liquid displacement method, as previously described ,24,25. B2 square areas. Young’s surface modulus was calculated based on force-displacement curves.Mechanical testing: The scaffolds for compressive strength and modulus measurement were prepared in 24-well plates with the freezing setting described above. Scaffolds were hydrated with PBS and trimmed into cylindrical shapes of 10 mm in height and 14 mm in diameter. The compression test was conducted at room temperature using a Shimadzu universal tester  with a rate of 0.4 mm/min until at least 50% strain was obtained. The compressive strength was determined as the compressive stress at the yield point. The compressive modulus was determined as the slope of the linear region of the stress–strain curve. The scaffolds for Young’s surface modulus measurement were sectioned into 400 µm thin slices and hydrated with PBS prior to measurement. Young´s surface moduli of scaffolds were obtained by conducting the nanomechanical measurement using a EasyScan atomic force microscope . The measurement was performed under an aqueous environment, using a ContAl-G  silicon nitride tip in a contacting mode. Each sample was scanned in an 8 × 8 array at three different 1 nmZeta potential measurement: The zeta potential of scaffolds was analyzed using a SurPASS 3 electrokinetic analyzer . Briefly, scaffolds were cast and freeze-dried into 1 mm thick sheets in 60 mm Petri dishes. The scaffolds were then neutralized, rinsed and trimmed to fit the size of the sample tube of the electrokinetic analyzer. The zeta potentials of the scaffolds were measured in triplicate using 0.01 M KCl as a buffer solution at pH = 7.4 to reflect the surface charge in physiological conditions.The human glioblastoma cell lines U-87 MG and U-118 MG were purchased from American Type Culture Collection . The human glioblastoma cell GBM6 was previously established in our laboratory ,27,28. Bv/v Calcien AM and 0.1% v/v PI), and then incubated for 30 min before imaging. Next, the scaffolds were mounted to microscope slides. A total of 10 μL of fluorescence dyes were added to prevent scaffolds from drying and covered with the coverslips immediately. Fluorescence images were captured using a Nikon TE300  inverted microscope.The cell morphology in scaffolds with different pore sizes was studied by observing the shape of tumor spheroid using live/dead imaging. The scaffolds were sectioned into 400 µm slices for better transparency prior to cell seeding. Cells were seeded on CHA scaffolds in a 96-well plate at 5000 cells per well, and cultured for 7 days in a fully supplemented medium. After Day 7, the fully supplemented medium was replaced by fluorescence dyes , following the manufacturer’s protocols. The reverse transcription was conducted using the iScript cDNA synthesis kit , following the manufacturer’s protocols to produce cDNA. Thermocycling was performed in a 20 μL solution system with 10 μL of SYBR Superrmix , 2 μL of 10 nM primers, 7 μL of DNase-free water and 1 μL of 50 ng/μL cDNA. The qRT-PCR was conducted on a CFX96 Touch Real-Time PCR Detection System . The thermocycle was set at 95 °C for 2 min, 40 cycles at 95 °C for 15 s, 58 °C for 30 s, and 72 °C for 30 s. Data were analyzed with the CFX Manager software  with expression levels normalized to GAPDH. The primers  are listed in v/v DMSO), 10, 50, 100, 200, 500, 1000, 2000 μM and treated for 72 h. After 72 h, the cell metabolic activities were measured by the AlamarBlue assay, as previously described. The relative cell viability was determined as the relative metabolic activities of the untreated control groups.Cells were seeded on 2D 96-well plates and CHA scaffolds in 96-well plates at 5000 cells per well and cultured in a fully supplemented medium for 7 days. After Day 7, the fully supplemented medium was replaced by a TMZ solution with a gradient concentration of 0 (with 0.1% v/v DMSO) as the negative control for 72 h. After 72 h, the cell metabolic activities were measured by the AlamarBlue assay, as previously described. The cell metabolic activities quantified by fluorescence intensities were used to calculate the Z′ factor and CV. The CV and Z′ for each cell line were obtained from three independent micro-plates. The CV was calculated by dividing the standard deviation of fluorescence intensity with average intensity. The Z′ was calculated using Equation (2).pσ and pμ are defined as the standard deviation and mean of the fluorescence intensity in positive controls. nσ and nμ are defined as standard deviation and mean of the fluorescence intensity in negative controls.The screening window coefficient, Z′ factor, was calculated to evaluate the quality of the CHA scaffolds as an HTS platform. The coefficient of variance, CV, was also calculated to analyze the result consistency. The CHA scaffolds with medium pore sizes were used for HTS validation. Briefly, cells were seeded on 2D 96-well plates and 8% CHA scaffolds in 96-well plates at 5000 cells per well and cultured in a fully supplemented medium for 7 days. After Day 7, cells were treated with 2000 μM TMZ solution as the positive control, and 0 μM TMZ .All data were presented as the mean ± standard deviation (SD) of the mean. ® or GeltrexTM. Therefore, it holds great promise for economic 3D HTS platform manufacture. The different scaffold pore sizes were achieved by altering the freezing-annealing time  was calculated to quantify the drug resistance.Here, the drug resistance of the three GBM cell lines was further analyzed based on the cell viability under a dose-dependent drug response test. Three cell lines were cultured in three CHA scaffolds, and tested using a standard anti-cancer drug for GBM therapy, temozolomide (TMZ) ,52. The Similar to the gene expression results, the three cell lines had distinct cellular responses to TMZ in scaffolds with different pore sizes. As shown in The criteria for assessing the quality of an HTS platform usually refers to two parameters: the cellular response uniformity and the drug screening window on 96- or 384-well-plate-based drug screening ,54. The In this study, we used a self-designed rapid dispensing system to achieve a fast and cost-effective large-batch production of 3D CHA porous scaffolds as a HTS platform. The 3D CHA porous scaffolds fabricated with adjustable pore size using a freeze-drying technique showed a significant difference in pore dimension, and a similar surface charge and micromechanical environment. We tested three GBM cell lines on the CHA scaffolds with different pore sizes and investigated the effect of pore size on the cell proliferation, morphology, and gene expression. Significantly, we performed a dose-dependent drug treatment using TMZ on the three cell lines in CHA scaffolds with varying pore size, and discovered that the results collectively capture the intertumoral heterogeneity of drug resistance. The heterogeneous nature of tumors is a major challenge in drug discovery and clinical trials. Our tunable HTS platform holds the potential of improving drug screening outcomes for different cancer cell lines by allowing us to adjust the pore size to achieve the highest drug resistance. In addition, we have demonstrated the high quality of the 3D HTS platform by validating comparable CV and Z′ factor values of three GBM cell lines tested with TMZ on CHA scaffolds and commercial tissue culture plates. Therefore, our 3D CHA-scaffold-based HTS platform stands as a powerful tool for future cancer research and new anti-cancer therapeutic development.


Here, we describe a patient with post‐transplant myelofibrosis with chronic graft‐versus‐host disease (GVHD), who showed successful molecular remission with ropeginterferon with 100% donor chimerism without any flare up of GVHD. He was initially diagnosed with polycythemia vera (PV) which progressed to myelofibrosis after 6 years. The MYSEC (Myelofibrosis Secondary to PV and ET‐Prognostic Model) and MTSS (myelofibrosis transplant scoring system) scores were 13.1 and 4, respectively, and the patient was in intermediate risk group. He underwent an allogenic stem cell transplant; however, his disease gradually progressed and was administered two donor lymphocyte infusions with minimal response. A second allogeneic transplant was performed, which led to a persistent molecular remission for more than a decade, although he developed chronic skin graft‐versus‐host disease (GVHD). The JAK2 V617F levels started to increase 10 years post‐transplant with ongoing chronic GVHD and a corresponding decrease in donor chimerism levels. He was administered ropeginterferon, which led to a decrease in JAK 2617F to undetectable levels. A graft versus myelofibrosis effect was attained with reversal to 100% donor chimerism, and he has since maintained a molecular remission with undetectable JAK 2617F levels. Chronic GVHD made him ineligible for donor lymphocyte infusions later. Thus, ropeginterferon was successful in inducing graft versus myelofibrosis effect, leading to a molecular response with no flare up of GVHD. The use of ropeginterferon needs to be further evaluated in larger cohorts of post‐transplant myelofibrosis patients. JAK2 level and chimerism after ropeginterferon treatment for relapsed myelofibrosis after allogeneic stem cell transplantation. Various strategies have been tried in different clinical settings to achieve a GVL effect. Interferons have been in use for a long time in different malignancies; however, its side effects have led to decreased compliance over time. However, the availability of newer pegylated versions has made its use more convenient.Here we discuss a case of relapsed post‐transplant secondary myelofibrosis, with chronic GVHD, successfully treated with ropeginterferon. The patient achieved a graft versus myelofibrosis effect with 100% reversal to donor cell chimerism and complete molecular remission without flare up of GVHD after using ropeginterferon.248‐year‐old man initially presented to our institute in the year 2000 with a persistently raised hemoglobin (Hb) and hematocrit values for few months.9/L. Serum LDH (lactate dehydrogenase) was normal.Clinical examination revealed nontender mild hepatosplenomegaly. The peripheral blood counts revealed the following: Hb 23.5 g/L; hematocrit, 0.69; white blood cell count (WBC), 10.8 × 10 *9/L; neutrophils, 79%; lymphocytes, 10%; eosinophils, 2%; basophils, 1%; monocytes, 8%; platelets, 272 × 10Abdomen ultrasound revealed homogenous splenomegaly with a spleen size of 17 cm × 5.9 cm. The vertical dimension of the liver in the midclavicular line was 17.2 cm. There was no evidence of any lymphadenopathy.Bone marrow evaluation revealed a markedly hypercellular marrow, with pan myeloid hyperplasia and dominating hematopoietic hyperplasia. Overall findings were consistent with a diagnosis of polycythemia vera rubra.Cytogenetics revealed trisomy 1 and the patient was positive for JAK‐2 V617 F mutation. He was initially prescribed hydroxyurea and underwent venesections, which he well tolerated.Six years after the initial diagnosis of PV, in 2006, he presented with generalized worsening tiredness, night sweats, and weight loss of 3–4 Kg in the last few months. Clinically, his splenomegaly had progressed, and the spleen measured 15 cm below the left costal margin, almost pressing onto the left kidney. This was accompanied by an increase in bone marrow fibrosis. Thus, his disease had progressed to post PV‐myelofibrosis.9/L; platelets 487 × 109/L; LDH, 609 U/L.The MYSEC score (Myelofibrosis Secondary to PV and ET‐Prognostic Model) of 13.1 . Put him in the intermediate 1 group, but with worsening splenomegaly and B symptoms.Blood reports showed the following: Hb, 15.7 g/L; WBC, 24.0 × 10In 2006, he underwent his first HLA (human leucocyte antigen) unrelated (9/10 HLA matched) donor stem cell transplant.The conditioning regimen used was fludarabine, busulfan, and antithymocyte globulin (ATG) according to EBMT protocol.However, he had residual disease, which gradually progressed. A year later, in 2007, he underwent two donor lymphocyte infusions (DLI) 2 months apart with 1 × 10*7 CD3+ cells/kg; however, this produced no significant response as well as no GVHD.In 2008, he underwent a second HLA matched unrelated alternative donor transplant after conditioning with treosulfan, fludarabine, and ATG. This time he went into persistent molecular remission and remained in remission for a long time.However, simultaneously he developed acute gut GVHD, which initially responded to steroids. This later developed into chronic GVHD of gut and skin, and he underwent extra corporeal photopheresis (ECP) to which he partially responded.He, however, continued to be in persistent complete remission. His JAK2 V617F levels were undetectable, and he maintained a full donor chimerism.His disease remained in control for more than a decade, until in 2019 when his chimerism studies revealed a slight downward trend to 98.9% along with chronic moderate skin GVHD. The JAK2 V617F levels kept increasing correspondingly and increased up to 4.13 from being undetectable. Donor lymphocyte infusion was not indicated due to persistent chronic GVHD.He was administered ropeginterferon 250 ug subcutaneously once every 2 weeks. He tolerated this well without any significant side effects for almost a year (September 2019–November 2020) and then discontinued it. He did not demonstrate any major autoimmune side effects, depression, any other significant cytopenia, or worsening of GVHD. Serial studies of both chimerism and JAK2 V617F were done. His chimerism studies gradually improved with the present study showing full chimerism. His JAK2 V617F levels became undetectable in January 2021.Figure At present, the patient is not on interferon and is clinically asymptomatic with stable blood reports.This case shows that interferon can produce a graft versus myelofibrosis effect in patients with relapsed myelofibrosis after allogeneic stem cell transplantation without worsening GVHD.3Interferons were introduced into usage a long time ago due to their innate ability to produce resistance to viral replication. Type I and 2 interferons act on human interferon alpha receptor chains and activate signaling through the JAK/STAT pathway. Interferons can also enhance natural killer and T cells, macrophage activity, and enhance expression of tumor associated and major histocompatibility complex (MHC) antigens.Interferon has been used in myeloproliferative neoplasms for a long time. Its use in polycythaemia vera (PV) and essential thrombocytosis (ET) has led to a decrease in mutant alleles of JAK2 V617F and calreticulin (CARL) and this has led to a renewed interest in expanding its usage. Interferon can suppress clonal hematopoiesis in essential thrombocytosis and polycythaemia vera. Prior to tyrosine kinase inhibitors, interferons have been used in chronic myeloid leukemia (CML) and has induced cytogenetic remission.The PEGINVERA study also demonstrates the effectiveness of ropeginterferon in achieving 80% complete hematological and molecular response in patients with long‐term usage.Molecular landscape consists of JAK2 V617F, CARL, MPL, or in their absence other clonal driver mutations .Till now, allogenic stem cell transplant remains the only potential curative treatment of myelofibrosis. This case described here is of post‐transplant secondary myelofibrosis, where the patient had a molecular relapse with ongoing chronic skin GVHD. He was administered ropeginterferon which was tolerated well without any flare up of his GVHD. Serial monitoring of his JAK2 V617F levels and chimerism studies revealed an excellent sustained response to treatment. He was initially diagnosed with polycythaemia vera, JAK2 V617F positive, which progressed to secondary myelofibrosis. He had minimal response to the first allogeneic stem cell transplant and donor lymphocyte infusions was administered. The disease, however, progressed, and he proceeded on to a second allogeneic transplant. He gradually developed acute followed by chronic skin and gut GVHD for which he was administered steroids and received ECP , and he maintained his remission for a long time.A follow up after 11 years revealed a drop in chimerism and a rise in JAK2 V617F levels. However, he had ongoing chronic skin GVHD. He was administered ropeginterferon at a dose of 250 ug subcutaneously every fortnight, which continued for a year. This led to a gradual rise in chimerism and decrease in JAK2 V617F levels. His last reports (February 2022) revealed chimerism of 99.9%, and JAK2 levels of 0.006. Interestingly, his skin GVHD also responded well and did not show any flare up.JAK2 V617F allele frequency; however, this effect in advanced myelofibrosis, if any, is small. The possibility of the direct tumor effect of interferon leading to the remission in our patients cannot be ruled out. However, as seen in patients with relapsed CML after allograft, the major effect of interferon postallograft is to increase HLA restricted antigen presentation, which allows better donor T‐cell mediated graft versus leukemia effect.It has been observed that better responses were seen in non‐T depleted transplants highlighting the importance of donor T cells to produce a DLI effect in controlling the malignancy. In PV, interferon is known to cause a reduction in We have used the term graft versus myelofibrosis effect here as our patient is a case of advanced myelofibrosis.As reported by Steegman et al, interferon has produced durable, complete cytogenic response with acceptable toxicity in relapsed patients with postallogenic transplant CML.Similar studies have also shown that interferon (ropeginterferon) is quite effective in producing cytogenetic remission in relapse post‐transplant allogeneic transplant patients of CML.This emphasizes the potential of interferons in immunomodulating effect and maintaining a sustained remission in myeloproliferative neoplasms. This line of treatment can be considered in patients with persistent residual disease or relapse after allogenic stem cell transplant to increase a graft versus myelofibrosis/leukemia effect, especially if donor lymphocyte infusions are contraindicated due to GVHD.4Interferons have been in use for a long time. However, due to the side effects of the initial preparation and the access to newer molecules its use has transiently decreased. However, with the newer pegylated preparations, there has been an increase in its use in hematological malignancies. With careful considerations to the known side effects, it can be used in patients with relapsed myelofibrosis after allogeneic stem cell transplantation when DLI is practically not possible.The use of interferon in producing graft versus myelofibrosis needs to be explored in a larger cohort of myelofibrosis patients relapsing after allogeneic stem cell transplant.Barnali Srivastava: Conceptualization; data curation; formal analysis; project administration; resources; visualization; writing – original draft; writing – review and editing. Christine Wolschke: Conceptualization; data curation; methodology; resources. Nico Gagelmann: Conceptualization; data curation; formal analysis; resources. Anita Badbaran: Conceptualization; data curation; formal analysis; resources. Nicolaus Kröger: Conceptualization; data curation; formal analysis; project administration; resources; software; supervision; visualization.The authors declare that they have no conflict of interest.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.The new aspect shown in this study is the efficient use of ropeginterferon in inducing a 100% chimerism in relapsed post‐transplant myelofibrosis. The central finding of the study is the induction of graft versus myelofibrosis effect with ropeginterferon and attaining a successful molecular remission in relapsed post‐transplant myelofibrosis cases ineligible for donor lymphocyte infusions. The specific clinical relevance of the study is considering the use of ropeginterferon to produce molecular remissions and induce 100% chimerism in patients with relapsed post‐transplant myelofibrosis.