Synovial sarcoma is a soft-tissue sarcoma that lacks effective systemic therapy and carries poor prognosis due to frequent late local recurrence and metastases. The cancer is known to be driven in part by increased expression of the pro-survival protein BCL-2. Surprisingly, synovial sarcoma proved resistant to BCL-2 inhibitors in pre-clinical trials. We identified increased activity of a second pro-survival protein, MCL-1, as responsible for this resistance. We showed that co-targeting both BCL-2 and MCL-1 proves to be an effective therapeutic approach both in cell culture and animal models of synovial sarcoma, supporting translation into clinical trials.Synovial sarcoma (SS) is frequently diagnosed in teenagers and young adults and continues to be treated with polychemotherapy with variable success. The SS18-SSX gene fusion is pathognomonic for the disease, and high expression of the anti-apoptotic BCL-2 pathologically supports the diagnosis. As the oncogenic SS18-SSX fusion gene itself is not druggable, BCL-2 inhibitor-based therapies are an appealing therapeutic opportunity. Venetoclax, an FDA-approved BCL-2 inhibitor that is revolutionizing care in some BCL-2-expressing hematological cancers, affords an intriguing therapeutic possibility to treat SS. In addition, there are now dozens of venetoclax-based combination therapies in clinical trials in hematological cancers, attributing to the limited toxicity of venetoclax. However, preclinical studies of venetoclax in SS have demonstrated an unexpected ineffectiveness. In this study, we analyzed the response of SS to venetoclax and the underlying BCL-2 family biology in an effort to understand venetoclax treatment failure and find a therapeutic strategy to sensitize SS to venetoclax. We found remarkably depressed levels of the endogenous MCL-1 inhibitor, NOXA, in SS compared to other sarcomas. Expressing NOXA led to sensitization to venetoclax, as did the addition of the MCL-1 BH3 mimetic, S63845. Importantly, the venetoclax/S63845 combination induced tumor regressions in SS patient-derived xenograft (PDX) models. As a very close analog of S63845 (S64315) is now in clinical trials with venetoclax in AML (NCT03672695), the combination of MCL-1 BH3 mimetics and venetoclax should be considered for SS patients as a new therapy. Synovial sarcoma (SS) is a rare but deadly soft-tissue sarcoma (STS) with no targeted therapy options. SS accounts for only about 10% of STS, but despite its relative rarity and predominant incidence in pediatric and young adults, 4000 cases are fatal each year . StandarL, and MCL-1), BH3-only pro-death , and terminal-effector molecules (BAK and BAX), the latter of which creates mitochondrial pores upon activation and oligomerization [L, and MCL-1 via BH3 mimetic drugs, which free BH3-activator proteins to initiate apoptosis, has been a major goal in pharmaceutical development [The BCL-2 family is a highly conserved family of proteins that functions primarily in and around the mitochondria, involving pro-survival , AMG 397 (NCT03465540), AZD-5991, and an inhibitor closely related to S63845 . The relative tolerability of venetoclax makes it an attractive agent to include in combination therapies, and clinical trials exploring concomitant dosing of MCL-1 inhibitors plus venetoclax are underway in several hematologic malignancies ,19. SS.PDX and SYO.1 cell lines were cultured in DMEM  with 10% FBS  and 1 µg/mL penicillin and streptomycin. SW982, ASKA, HS-SY-II, and Yamato cells were grown in RPMI1640  and 1 µg/mL penicillin and streptomycin. Routine mycoplasma testing was performed on all cell lines. The SS.PDX cell line was established ex vivo from the SS patient-derived xenograft model. SYO.1 and SW982 cell lines were kindly provided by Cyril Benes and the Center for Molecular Therapeutics at Massachusetts General Hospital . ASKA, HS-SY-II, and Yamato cell lines were obtained from Riken Bioresource Center . http://hgserver1.amc.nl, accessed on September 2019) [RNA expression data for sarcoma samples were obtained and analyzed through the R2: Genomic Analysis and Visualization Platform. (er 2019) . L (2764), BIM (2933), NOXA (14766), and SS18 (70929) from Cell Signaling Technology . Secondary antibodies used were anti-mouse IgG  and anti-rabbit IgG . S63845 was from Abmole , and venetoclax (ABT-199) was kindly provided by AbbVie Inc . Primary antibodies used for western blotting were as follows: GAPDH (sc-3233) and MCL-1 (s-19) from Santa Cruz Biotechnology; cleaved PARP1 (5625), cleaved Caspase III (9579), BCL-2 (4223), BCL-X6 cells were injected s.c. into each flank of 6–8-week-old NOD/SCID gamma (NSG) mice. The patient-derived xenograft model was acquired from Jackson Laboratories (J000104314). For the SYO.1 xenograft model, 5.0 × 106 cells were injected s.c. into each flank of 6–8-week-old female NOD/SCID gamma (NSG) mice. Treatment began when tumors reached approximately 150 to 200 mm3, as calculated using the L*W2 *.52 method. Mice were randomized into treatment cohorts, and tumor size was measured at least three times per week for the duration of treatment. Venetoclax was dissolved in 60% Phosal 50 PG, 30% PEG400, and 10% ethanol for a final dosage of 100 mg/kg body weight and administered via oral gavage five days a week [For the SS patient-derived xenograft model, 5.0 × 10s a week ,22. Nexts a week ,15. Then3 cells/well in 180 µL media. After 24 h, cells were treated with increasing concentrations of ABT-199 and S63845, alone or in combination, for 72 h, at which point 25 µL CTG reagent was added to each well, and plates were read using a Synergy H1 Hybrid microplate reader . These experiments were each repeated in triplicate over the course of several weeks. For crystal violet assays, 5.0 × 104 cells were seeded in 6-well plates for 24 h and then treated with ABT-199 and S63845, alone and in combination. Media was changed every other day until the untreated well was fully confluent, at which point cells were stained with 0.1% crystal violet . For FACS measurements of apoptosis, 2.0 × 105 cells were seeded in 6-well plates and after 24 h, were treated with ABT-199 and S63845 as indicated. After 24 h, cells were collected per BD Biosciences Annexin V/PI staining protocol and then assayed on a Millipore Guava flow cytometer. FACS experiments were conducted in biological triplicate. For CellTiter-Glo (CTG)  assays, cells were seeded in quadruplicate in 96-well plates at a concentration of 1.5 × 10NOXA overexpressing SS.PDX and SYO.1 cell lines were generated as previously described . In briet-test comparisons were performed using R2: Genomics Analysis and Visualization Platform . For the in vivo experiments, Unpaired two-tailed t-test comparing tumor volumes, and paired two-tailed t-test in comparing mouse weights, were performed using GraphPad Prism version 8.4.2 for macOS . For CTG experiments, the Bliss additivity method was used to calculate synergy scores [For RNA expression data, one-way ANOVA followed by two-tailed y scores .L) or pro-apoptotic proteins  can alter sensitivity to BCL-2 inhibition [L (BCL2L1) expression trended lower in the SS tumors, suggesting it is not a critical factor in venetoclax resistance [Despite high BCL-2 expression being diagnostic for SS, BCL-2 targeting has proven to be an ineffective strategy in preclinical SS models . As the hibition ,25,26,27hibition . As expesistance . In contL and MCL-1 were largely unchanged across cell lines with either construct. We next analyzed changes in BCL-2 family members after endogenous expression of SS18-SSX in a normal human fibroblast line, CRL-7250 [The SS18-SSX fusion protein can both activate and repress target gene transcription . The fusCRL-7250 . ConsistCRL-7250 ,31,32,33As low NOXA expression has been demonstrated to mediate venetoclax resistance in hematologic cancers ,34, we hAfter observing that NOXA expression sensitized SS cell lines to venetoclax and S63845 similarly sensitized SS cell lines, we chose to expand our investigation into the efficacy of venetoclax in combination with S63845 as a therapy for SS. We exposed a panel of SS cell lines to venetoclax and S63845, both alone and in combination. After 72 h of treatment, cell viability was assessed using CTG assay. Each drug showed limited efficacy as a single agent, yet strong synergistic cell killing in combination across all five cell lines expressing the SS18-SSX fusion gene A. In conWe also conducted seven-day treatments to assess SS response to the combined treatment B. BecausBCL-2 and MCL-1 are critical anti-apoptotic proteins and BH3 mimetics against these two molecules that induce apoptosis ,35. We fThese data prompted further investigation into how venetoclax and S63845 interacted to increase the amount of apoptosis. BIM is a pro-apoptotic protein that mediates venetoclax-mediated cell killing ,21. AnalWe next assessed the anti-tumor effect of venetoclax and S63845 in a patient-derived xenograft (PDX) model of SS. Mice were treated with 100 mg/kg of venetoclax  delivereSS accounts for ~10% of pediatric STS and, due to its metastatic potential and the very limited efficacy of chemotherapies, is often fatal . The knoVenetoclax is effective in the clinic as a monotherapy against chronic lymphocytic leukemia (CLL), which expresses BCL-2 at high levels , as wellMCL-1 is among the most well-characterized resistance factors for venetoclax. As a structurally related anti-apoptotic BCL-2 family member, MCL-1 can bind to pro-apoptotic factors released from BCL-2, such as BIM and BAK, thus preventing venetoclax-mediated apoptosis ,43,44. NSS has long been known to characteristically overexpress BCL-2 ; SS18-SSMultiple MCL-1 inhibitors are now being evaluated in early clinical trials . In addiDespite high levels of BCL-2 in SS, the activity of venetoclax in preclinical models of SS has been disappointing. This study demonstrates that suppressed NOXA expression leads to an MCL-1-mediated resistance to venetoclax in SS, thus revealing an Achilles’ heel that can be exploited by concomitant treatment with an MCL-1 inhibitor. Simultaneous disruption of both BCL-2 and MCL-1 via small-molecule inhibitors induces robust regression of tumors in a SS PDX model and cell-line xenograft model as well as multiple cell lines derived from both primary and metastatic disease. Given the lack of therapeutic options for SS, these data provide the rationale for clinical evaluation of this combination.


Clinical trials have evidenced that several natural compounds, belonging to the phytochemical classes of alkaloids, terpenes, phenols and flavonoids, are effective for the management of various types of cancer. Latest research has proven that natural products and their semisynthetic variants may serve as a starting point for new drug candidates with a diversity of biological and pharmacological activities, designed to improve bioavailability, overcome cellular resistance, and enhance therapeutic efficacy. This review was designed to bring an update regarding the anticancer potential of betulonic acid and its semisynthetic derivatives. Chemical derivative structures of betulonic acid including amide, thiol, and piperidine groups, exert an amplification of the in vitro anticancer potential of betulonic acid. With the need for more mechanistic and in vivo data, some derivatives of betulonic acids may represent promising anticancer agents. The extensive research over the past decades has identified an increased number of natural compounds from various medicinal plants that elicit chemopreventive potential. These phytochemicals represent an important potential source of anticancer molecules as such or after various physico-chemical modulations, which can be successfully used in different therapeutic protocols ,2. The aCancer is one of the largest causes of mortality in the world and due to its prevalence, the discovery of novel anticancer drugs has great importance. Plants, microorganisms, and marine organisms are considered valuable sources for the discovery of anticancer drugs ,5.Throughout history, natural products and their derivatives have proved to be useful for the development of chemotherapeutics, presenting a great structural diversity as well as molecular and pharmacological properties that favor such development .Vinca rosea L. (Apocynaceae), were the first agents to advance into clinical use. At low doses, they interrupt microtubular activity, whereas, at higher doses, they cause cell cycle arrest and apoptosis  in betulin)  = 4.2–32.0 µg/mL), as shown in 2)n in the amide residue did not affect the inhibitory activity, however, the introduction of the second amide residue increased the inhibition of the tumor cell growth (I (D)). The apoptosis-inducing activity of betulonic acid amides was higher than that of betulonic acid for MT-4 and MOLT-4 cell lines. Thus, the introduction of amide function at the position C28 of PT led to the formation of effective inducers of apoptosis methyl}-3-oxolup- 20(29)en-28-oate (XXI) were also examined in terms of pro-apoptotic activity; they induced DNA fragmentation in colon cancer cell line SW480, thereby providing strong signs of apoptosis. In addition, acridine orange/ethidium bromide staining of A549 lung carcinoma cells treated with these derivatives also showed induced apoptosis [The pro-apoptotic and cytotoxic activity of betulonic acid derivatives was reported by Csuk et al. The research groups have synthesized C(2)-methylene derivatives either by Mannich reactions, accompanied by poptosis .Kazakova et al. have reported the biological activity of C-28-imidazolides of betulonic acid, containing 3-oxo-, 3-hydroxyimino-, and 2-cyano-2,3-seco-4(23)-ene fragments in cycle A. Firstly, they assessed the cytotoxicity of the imidazolides (cycle A) using different human tumor cell lines , dyed with sulforhodamine B, and treated with 10 μM of the derivatives for 48 h. The results have shown that the imidazolide of 3-hydroxyimino-lup-20(29)-en-28-carboxylic acid (cycle A) (XXII) was the most efficient in inhibiting the cell growth of all six lung cancer cell lines, all seven large intestine cancer cell lines, all six leukemia cell lines, all six melanoma cell lines, all four breast cancer cell lines, two nervous system cancer cell lines, one prostate cancer cell line, three ovarian cancer cell lines, and five renal cancer cell lines. The cell survival rates are depicted in Owing to the hydrophobic properties of betulonic acid, its in vivo anti-cancer effectiveness has not been intensively studied . In a reBased on the remarkable results obtained for the imidazolide of 3-hydroxyimino-lup-20(29)-en-28-carboxylic acid (XXII), Kazakova et al. have studied its antineoplastic activity in vivo on two ascites tumors (Ehrlich tumor and P388 lympholeukemia) and three solid tumors  inoculated in BDF1 hybrids (DBA2 × C57B1/6J), BALB/C, and DBA mice. This chemical derivative injected intraperitoneally, five times daily at a dosage of 70 mg/kg, had no antineoplastic effect neither on the Ehrlich ascites tumor nor P388 lympholeukemia (at a dose of 30 and 50 mg/kg), respectively. Interestingly, the administration of 50 mg/kg through the same procedure suppressed the development of breast adenocarcinoma (Ca755) by 56–77%. The result was maintained without an improvement in life duration until day eight of the study. In mice with large intestinal adenocarcinoma treated by the same procedure, a 53% decrease in tumor growth was seen only after 5 days of therapy without any change in the mice’s lifespan .The research study conducted by Zhukova et al. examined the effects of betulonic acid and its methyl esters derivatives on the pathological modifications in the kidneys of the C57BL/6 mice, presenting Lewis pulmonary adenocarcinoma. Compounds were administered intraperitoneally in a dosage of 50 mg/kg for 8 days. After 8 days, mice were decapitated under ethereal anesthesia and sections of the treated kidneys were analyzed using periodic acid–Schiff reaction-hematoxylin and eosin-orange G dyeing. These chemical derivatives had a beneficial impact on the course of paraneoplastic nephropathy; they decreased the volume density of epithelial cell necrosis at 56%. On average, the number of cells with edematic cytoplasm, vesicular lipid infiltrate, and disrupted brush lymbus fell by 8–13% in comparison to untreated tumoral cells .Data presented in this work show that following structural modifications, which involved the introduction of amide, thiol, and piperidine groups, amplification of the in vitro anticancer potential of betulonic acid can be achieved. Studies published in the state of the art literature revealed that betulonic acid derivatives possess an important in vitro cytotoxic and pro-apoptotic activity. However, the number of in vivo studies is limited. The screening of scientific literature on this topic indicates a demand for in-depth in vivo data regarding the effect and mechanism of action of the betulonic acid derivatives in order to benefit from these structures in different therapeutic protocols designated for the management of various types of cancer.


Single crystal X-ray diffraction analysis of 1–3 and a dimethylsulfoxide (DMSO) solvate of 2 (2-DMSO), has identified the presence of a range of intra- and intermolecular interactions including N-H⋯N, N-H⋯O=C and N-H⋯O=S(CH3)2 hydrogen-bonding interactions, C-H⋯π interactions and off-set, face-to-face stacking π-π interactions that support the variety of slipped stack, herringbone and cofacial crystal packing arrangements observed in 1–3. Additionally, the cytotoxicity of this series of aromatic oligoamides was assessed against two human ovarian (A2780 and A2780cisR), two human breast (MCF-7 and MDA-MB-231) cancerous cell lines and one non-malignant human epithelial cell line (PNT-2), to investigate the influence of the terminal functionality of these aromatic oligoamides on their biological activity. The chemosensitivity results highlight that modification of the terminal group from Ac to Boc in 1 and 2 leads to a 3-fold increase in antiproliferative activity against the cisplatin-sensitive ovarian carcinoma cell line, A2780. The presence of the amine termini in 3 gave the only member of the series to display activity against the cisplatin-resistance ovarian carcinoma cell line, A2780cisR. Compound 2 is the lead candidate of this series, displaying high selectivity towards A2780 cancer cells when compared to non-malignant PNT-2 cells, with a selectivity index value >4.2. Importantly, this compound is more selective towards A2780 (cf. PNT-2) than the clinical platinum drugs oxaliplatin by > 2.6-fold and carboplatin by > 1.6-fold.The synthesis and characterization of three aromatic oligoamides, constructed from the same pyridyl carboxamide core but incorporating distinct end groups of acetyl (Ac)  The rise of cancer cell resistance towards clinical anticancer drugs, combined with the poor selectivity they can demonstrate for cancers over non-malignant tissue and the occurrence of adverse side-effects, has driven the search for new compounds with increased antiproliferative activity and selectivity. . Whilst 50 values (1.1–4.3 μM) against the human colon carcinoma (DLD-1), mesothelioma (I45), lung carcinoma (A549), and human non-small cell lung carcinoma (H1299).Systematic solid-state studies of aromatic oligoamides have identified that small structural variations in these molecules can have a profound influence on their conformational behavior and such studies can to help deepen our understanding of their structure-activity relationships (SARs). A crystallographic study of aromatic oligoamides by Nissinen and co-workers  showed t1, tert-butyloxycarbonyl (Boc) 2 and amine 3 , carboplatin (CARB) and oxaplatin (OXA), which all demonstrate high cytotoxicity.Gaining an understanding of the influence of the structure of an aromatic oligoamide on its biological activity is central to the development of new molecules within this class that have the potential to demonstrate improved cytotoxicity towards cancerous cells. To probe the influence of the terminal group on the solid-state structure and antiproliferative activity of these aromatic oligoamides, we undertook the synthesis, crystallographic analysis and cytotoxicity studies of three aromatic oligoamides based on the same pyridyl carboxamide core but including different end groups; acetyl (Ac)  amine 3 . We repo1–3, which all have the same pyridyl carboxamide core, but incorporate different terminal groups of Ac 1, Boc 2 and NH23 . Electronspray ionization mass spectrometry identified the molecular ion peaks at m/z 432.1670 [M + H]+1, 548.2503 [M + H]+2 and 348.1453 [M + H]+3.Aromatic oligoamides, nd NH23  have beend NH23  . 1–3 and 2-DMSO . X-ray diffraction analysis identified the nature of the non-covalent interactions present in the solid state for each of the studied aromatic oligoamides.Single crystals suitable for X-ray diffraction were obtained for 1 were grown by vapor diffusion of diethyl ether into a dimethylformamide solution at ambient temperature. 1 crystallizes in a monoclinic crystal system and solution refinement was performed in the P21/c space group -H (2/4)⋯N (1) (2.6583(17)-3.2310(16) Å, 1 displays a slipped stack crystal packing arrangement,  solvate, 2-DMSO, were grown from two different crystallization conditions at ambient temperature, firstly, through the slow evaporation of chloroform to give 2 and secondly, through the slow evaporation of a 9:1 chloroform:DMSO solvent mixture to generate 2-DMSO. In the former conditions, 2 crystallizes in an orthorhombic crystal system and solution refinement was performed in the P212121 space group -H (2/4A)⋯N (1) 2.34(4)-2.36(4) Å, The molecular structures of 2 adopts a herringbone crystal packing arrangement -H (3)⋯O (4) = C and N (5)-H (5)⋯O (1) = C hydrogen bonding interactions, (2.03(4)-2.231(4) Å, tert-butylcarboxyaminophenyl rings on neighboring molecules -H (3)⋯O (4) = C, (2.012 (18) Å, 3)2 hydrogen-bonding interaction present which involves one of the NH’s of a terminal group moiety and the O atom of a DMSO solvent molecule (N (5)-H (5)⋯O (7) = S(CH3)2, (2.007 (19) Å, In the crystal packing of 3 were grown through the slow evaporation of chloroform at ambient temperature. 3 crystallizes in a monoclinic space group and solution refinement was performed in the P21/c space group -H (3B)⋯O (2) 2.15 (7) Å (3 (angement  orientat (c axis  and this15 (7) Å , N (8)-H15 (7) Å  and N (115 (7) Å . The sec15 (7) Å . Additio(7) Å (3 . (Egli e(7) Å (3 .CDDP), carboplatin (CARB) and oxaliplatin (OXA) and compounds 1–3 were screened for their cytotoxicity against human cell lines: cisplatin-sensitive ovarian carcinoma (A2780), cisplatin-resistant ovarian carcinoma (A2780cisR) and breast adenocarcinomas (MCF-7 and MDA-MB-231). The IC50 values were obtained via the MTT assay after a 96 h incubation period of each compound with the cells at 37°C and 5% CO2 . The amine-terminated compound 3 in non-toxic towards the breast adenocarcinomas cell lines (MCF-7 and MDA-MB-231), with IC50 values greater than the tested threshold (>100 μM). Notably, 3 is non-toxic against the cisplatin-sensitive ovarian carcinoma A2780 but is the only one in the library which displays any level of antiproliferative activity against the cisplatin-resistant ovarian carcinoma cell line, A2780cisR, with a moderate IC50 value of 61 ± 1 μM.Cisplatin  and breast adenocarcinomas (MCF-7 and MDA-MB-231). Results highlight the Boc-terminated compound CDDP, OXA, and CARB and compounds 1–3 were also screened against the non-malignant prostate cell line , PNT-2, to determine any cancer cell selectivity. The results for CDDP, OXA, and CARB show that these clinical platinum drugs have high to moderately cytotoxicity towards PNT-2, with IC50 values of 1.3 ± 0.2 μM (OXA), 8.5 ± 0.4 μM (CDDP) and 27 ± 2 μM (CARB). Unlike the clinical platinum drugs, compounds 1–3 are non-toxic towards PNT-2  The selectivity index (SI) values were calculated for all the compounds, using the IC50 values obtained against PNT-2 and dividing by the IC50 value against the cancer cell line in parenthesis in 1 shows only slight increases in selectivity, with an SI > 1.3*  for this compound against the triple negative breast cancer (TNBC) cell line, MDA-MB-231, is higher than those observed for the clinical platinum anticancer drugs OXA and CARB (of 0.5 and 0.8 respectively).1 Compound 2 displays a notable SI > 4.2* (p < 0.05) against A2780 and a very moderate increase in selectivity towards MCF-7  and MDA-MB-231 (SI > 1.4*).3, is the only compound to display increased selectivity for A2780cisR when compared to PNT-2, with a SI > 1.6* (p < 0.05), which, albeit is very modest, is higher than the SI values observed for CDDP (0.6), OXA (0.6) and CARB (0.3*). Overall, these results highlight that small structural changes to the terminal groups of these aromatic oligoamides can have a marked effect on their biological activity against the tested ovarian and breast cancer cell lines. Herein, it is shown that modification of the terminal groups from Ac to Boc leads to a notable increase in the SI against A2780 but similar SI values are observed against MDA-MD-231, whilst variation of the terminal group to NH2 leads to a change in the SI of the aromatic oligoamide against A2780cisR with a slight increase in SI  being observed by this amine-terminated compound.1, tert-butyloxycarbonyl (Boc) 2 and amine 3. Single crystal X-ray diffraction analysis of 1–3 and 2-DMSO has identified the presence of an array of non-covalent interactions including N-H⋯N and N-H⋯O=C hydrogen-bonding interactions, a series of C-H⋯π and π-π stacking interactions that support the diverse crystal packing arrangements present in these aromatic oligoamides including slipped stack (1), herringbone (2) and cofacial/slipped stacked (3). The crystal packing of 3 also reveals the presence of hydrogen-bonded dimer formed by the presence of reciprocal intermolecular N-H⋯O=C hydrogen bonding interactions formed between the NH of the terminal amine groups and the O atom on the carbonyl group in the amide group of an adjacent molecule.In conclusion, we have synthesized and characterized a series of aromatic oligoamides based on a common pyridyl carboxamide core but incorporating distinct end groups: acetyl (Ac) 1–3  were obtained via a 96 h MTT assay, and screening against human ovarian carcinomas (A2780 and A2780cisR), human breast adenocarcinomas (MCF-7 and MDA-MB-231) and non-malignant prostrate cell line (PNT-2). Generally, compounds 1–3 display either moderate cytotoxicity or are non-toxic against A2780cisR, MCF-7 and MDA-MB-231 cancer cell lines. The Boc-terminated compound, 2, is the lead candidate of the tested aromatic oligoamides displaying an IC50 value of 24 ± 0.9 μM against A2780. Unlike the tested clinical platinum anticancer drugs, compound 2 is non-toxic towards PNT-2 (IC50 > 100 µM), meaning it displays an SI value >4.2*-fold towards A2780 (cf. PNT-2), making it more selective towards ovarian cancer than the platinum drugs CDDP and OXA , 6.5 (CARB); 2.6 (OXA)). The insights gained from this study, regarding the importance of small structural modifications on influencing the biological activity of aromatic oligoamides, will facilitate the future design of related compounds with improved cytotoxicity against ovarian and breast cancer cell lines.To understand SARs, the cytotoxicity of the compound


Background: Systemic treatment options for metastatic renal cell carcinoma (RCC) have significantly expanded in recent years. However, patients refractory to tyrosine kinase and immune checkpoint inhibitors still have limited treatment options and patient-individualized approaches are largely missing. Patients and Methods: In vitro drug screening of tumor-derived short-term cultures obtained from seven patients with clear cell RCC was performed. For one patient, a patient-derived xenograft (PDX) mouse model was established for in vivo validation experiments. Drug effects were further investigated in established RCC cell lines. Results: The proteasome inhibitor carfilzomib was among the top hits identified in three of four patients in which an in vitro drug screening could be performed successfully. Carfilzomib also showed significant acute and long-term cytotoxicity in established RCC cell lines. The in vivo antitumoral activity of carfilzomib was confirmed in a same-patient PDX model. The cytotoxicity of carfilzomib was found to correlate with the level of accumulation of ubiquitinated proteins. Conclusions: In this proof-of-concept study, we show that patient-individualized in vitro drug screening and preclinical validation is feasible. However, the fact that carfilzomib failed to deliver a clinical benefit in RCC patients in a recent phase II trial unrelated to the present study underscores the complexities and limitations of this strategy. Metastatic renal cell carcinoma (RCC) has a poor prognosis with 5-year survival rates between 8% and 12% ,4,5. DesAdvanced RCC is characterized by a high degree of genomic and functional intratumoral heterogeneity ,7. UnderIn the present proof-of-concept study, we demonstrate the feasibility of simultaneous patient-individualized in vitro drug screening and PDX development in metastatic RCC. The potent proteasome inhibitor carfilzomib was identified as the most effective compound in vitro and its antitumoral activity was confirmed in vivo. In light of a negative phase II study of carfilzomib in patients with advanced RCC, published recently , complexl-glutamine . All experimental procedures were approved by the Ethics Committee of the University of Heidelberg School of Medicine (S-453/2010). Written informed consent was obtained from all patients.Short-term tumor cell cultures were established as previously described in . Brieflyhttps://dtp.cancer.gov/organization/dscb/obtaining/available_plates.htm (accessed on 21 November 2013). The compounds of the library included chemotherapeutic agents, small molecule inhibitors, and antihormonal substances. Briefly, tumor cells were seeded into 96-well plates with 10,000 cells per well and allowed to adhere overnight. Drugs were then added to the tumor cells at a final concentration of 1 and 10 μM, respectively. The drug concentrations used for hit discovery, i.e., 1 and 10 μM, were based on the published literature [VHL status [2.Short-term tumor cell cultures were subjected to a medium-throughput functional drug screening procedure using the National Cancer Institute’s (NCI) Developmental Therapeutics Program Approved Oncology Drugs Set IV. A full list of compounds can be found under L status . All celPrimary tumor cells were injected subcutaneously into the flanks of Naval Medical Research Institute (NMRI)-nude mutant mice. The growth of palpable tumors was monitored and followed by randomized treatment with the pharmacologic agents (vehicle control or 3 mg/kg/d carfilzomib i.v.). The treatment group consisted of five animals; the control group consisted of four animals. To determine therapeutic efficacy, tumors were measured twice weekly. The study was performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the Society of Laboratory Animals (GV SOLAS). All animal experiments were approved by the Committee on the Ethics of Animal Experiments of the Freiburg regional council  and performed at Charles River Laboratories, Freiburg, Germany.The Proteome Profiler™ Array for human ubiquitinated proteins was used as recommended by the manufacturer .t-test for independent samples was used wherever applicable. p values ≤ 0.05 were considered statistically significant.Student’s two-tailed Short-term tumor cell cultures from seven RCC patients were established and used for functional drug screening with the NCI Approved Oncology Drugs Set IV . For pat50 was reached), and only drug screens with at least one compound producing such a reduction of cell viability were considered successful in our analyses [A drug hit was defined as a ≥0.5-fold change in cell viability by a compound .We were able to further validate the result that carfilzomib is highly effective in RCC001 by testing short-term tumor cell cultures obtained from two distinct anatomic locations c,d. BothTaken together, functional drug screening of tumor-derived short-term cultures and validation in a same-patient PDX model demonstrate antitumoral effects of the proteasome inhibitor carfilzomib in advanced RCC.We next investigated whether carfilzomib has acute antitumoral activity in established RCC cell lines as well as non-cancerous, kidney-derived HEK293 cells. First, we tested the response to carfilzomib in these cell lines and found growth-inhibitory activities in all cell lines, starting at a concentration of 0.01 μM .VHL wildtype) and 786-0 cells (VHL mutated), with HEK293 cells used as controls, and analyzed the response to carfilzomib in comparison to sunitinib, sorafenib and axitinib, which are VEGF receptor targeting receptor tyrosine kinase inhibitors (TKIs) approved for the treatment of metastatic RCC  under normoxic conditions. In case of hypoxia, and when VHL is inactivated, HIFs accumulate and activate signaling pathways that lead, among others, to enhanced angiogenesis via the VEGF/VEGF receptor (VEGFR) axis. This pathway is the basis for established and novel therapeutic interventions in RCC that target VEGF, VEGFR or HIF2α [Unfortunately, in a recent phase II study of nine patients with treatment-refractory metastatic clear cell RCC, there was an absence of antineoplastic activity of carfilzomib . All ninionality . The VHLor HIF2α .In a situation, however, where destabilized pVHL with at least partially retained functionality is expressed, pVHL activity could potentially be rescued by inhibition of the proteasome with a subsequent re-activation of HIF degradation . Given tFirst, it is conceivable that short-term cultures and established cell lines do not adequately reflect the complex microenvironment in a patient tumor .Second, proteasome inhibition has been reported to attenuate immune cell functions . RCC is There are a number of additional points that need to be taken into account when patient-individualized drug screening and validation are considered. The screening set-up may have favored finding drugs like carfilzomib, since it is known for relatively fast apoptosis induction, while, for example, TKIs are more dependent on cell growth and inhibition of signal transduction pathways. In addition, the target structure of TKIs may not be expressed by the cells analyzed. As such, for drugs with different types of kinetics and mechanisms of action, different assays may be appropriate .It may be also possible to exploit the drug screening results for potential combination therapies. For example, in patient RCC006, carfilzomib and the EGFR-targeting TKI erlotinib were both found as hits . A previThe off-label use that almost inevitably follows drug screening efforts has legal ramifications that can prolong or prevent drug procurement. In addition, low engraftment rates and slow tumor growth can delay the generation of PDX models. Moreover, serial passaging has been shown to alter drug sensitivities and evolutionary trajectories away from the primary tumor ,38,39. IVHL mutation testing has not been performed. However, since the vast majority of patients with clear cell RCC harbor inactivated VHL, it is highly likely that this is also the case in our patients. We found a more pronounced accumulation of ubiquitinated proteins in a VHL-deficient RCC cell line pointing to a more general role of pVHL in the elimination of abnormal proteins, as previously suggested [Further limitations of our proof-of concept study are the small sample size and the lack of genetic information of the tumors. Unfortunately, uggested .Taken together, our results show that patient-individualized drug screening and parallel in vivo validation is feasible but at the same time underscore the complexity of this approach. Further studies are warranted to understand the apparent disconnection between preclinical and clinical findings and to optimize tools for drug discovery in advanced RCC.


Since, phosphatidyl-inositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) and fibroblast-growth-factor-receptor-3 (FGFR3) mutations often occur in such tumors, here, we tested targeted therapy directed to such genes in TSCC/BOTSCC cell lines. We also combined the two types of inhibitors with each other, and cisplatin or docetaxel that are used clinically.Human papillomavirus positive  approved drugs, alpelisib (BYL719) and erdafitinib (JNJ-42756493) alone and in combination with cisplatin or docetaxel. Viability, proliferation, apoptosis and cytotoxicity responses were thereafter followed by WST-1 assays and the IncuCyte S3 Live® Cell Analysis System.The HPV+ CU-OP-2 had a pS249C-FGFR3, and like CU-OP-20, a pE545K-PIK3CA mutation, while no other lines had such mutations. Irrespectively, dose dependent responses to all PI3K/FGFR inhibitors were obtained, and upon combining the inhibitors, positive effects were observed. Cisplatin and docetaxel also induced dose dependent responses, and upon combination with the inhibitors, both positive and neutral effects were found.HPVThe data suggest that FDA approved drugs alpelisib and erdafitinib efficiently inhibit TSCC/BOTSCC cell line growth, especially when combined irrespective of presence of corresponding mutations and should be further explored, for use upon recurrent disease. Stock solutions were at 10 mM for all inhibitors, except BEZ235 where a stock solution of 2 mM was recommended. Further dilutions were done in PBS, and were for: AZD4547 5-25 μM; JNJ-42756493 0.01-10 μM; BEZ235 0.25-1 μM; and for BYL719 0.5-20 μM. Cisplatin  was diluted in PBS and used at 2.5-7.5 μM. Docetaxel  was diluted in PBS and used at 0.6-6 nM. All experiments were repeated minimum three times.Viability was quantified using the WST-1 assay  and followed for 72 h according to the instructions of the manufacturer and before , 42.® Cell Analysis System, using the IncuCyte Caspase-3/7 Green Apoptosis Assay Reagent and the Incucyte™ Cytotox Red Reagent , and followed for proliferation, apoptosis and cytotoxicity by images taken every 2 h  decreased viability consistently (p<0.01) , while CU-OP-2 and -3 were more resistant , . For UPCI-SCC-154 and UT-SCC-60A only data on the FDA approved drugs are shown, since data on the others have been published  , for all remaining, at least p<0.05  of BYL719-JNJ-42756493 were calculated for all lines 24-72 h after treatment as done before , 42–44. To summarize, combining PI3K-FGFR inhibitors, generally resulted in positive combinatory effect in most cell lines.All cells lines presented dose dependent responses to single treatments with cisplatin or docetaxel as shown with WST-1 assays in All CU-OP lines presented decreased viability compared to PBS with 2.5-7.5 µM of cisplatin 72 h after treatment, except for CU-OP-20 with 5 µM cisplatin     of BYL719 and/or JNJ-42756493 with cisplatin or docetaxel were calculated for all cell lines 48-72 h after treatment , 38–40, To summarize, mainly positive and neutral effects were obtained upon combining BYL719 and JNJ-42756493 resp. with cisplatin and docetaxel on CU-OP lines, with the best effects observed for CU-OP-2 and CU-OP-17.Effects on proliferation, apoptosis and cytotoxicity by single and combined BYL719 and JNJ-42756493 treatments on the CU-OP lines were followed by the IncuCyte S3 Live−Cell Analysis System. All lines showed complete inhibition of proliferation with all BYL719 doses, and the highest JNJ-42756493 dose, and upon combined exposures excluding the highest doses of both inhibitors, proliferation was inhibited with all combination doses Figure 6Cytotoxicity was observed for all CU-OP lines with the two highest BYL719 (1 and 10 µM) and the highest JNJ-42756493 (10 µM) concentrations. Upon combined exposure, cytotoxicity was observed in most lines, especially those including 1 µM BYL719 .Combining BYL719 (0.5-10 µM) or JNJ-42756493 (0.01-1 µM) with either 7.5 µM cisplatin or 6 nM docetaxel, inhibited proliferation completely (data not shown). Cytotoxicity was observed with the two highest BYL719 (1 and 10 µM) and the highest JNJ-42756493 (10 µM) doses with cisplatin or docetaxel for CU-OP-2 and -3, while CU-OP-20 and CU-OP-17 were generally less sensitive especially when docetaxel was present with JNJ-42756493 (data not shown). Prominent apoptosis was only observed in combinations including the highest JNJ-42756493 dose (10 µM) (data not shown).In this study, the FDA approved drugs alpelisib and erdafitinib induced dose dependent effects with decreased viability and proliferation on HPV+ and HPV- TSCC/BOTSCC cell lines, irrespective of whether the cells had FGFR3 and/or PIK3CA mutations. Furthermore, upon combining these inhibitors mainly positive or neutral effects were disclosed with comparable decreases in viability and proliferation using lower doses of the inhibitors. All tested TSCC/BOTSCC cell lines above also presented dose-dependent effects with diminished viability and proliferation when treated with cisplatin and docetaxel often used clinically, although e.g., CU-OP-2 and CU-OP-3 were relatively more resistant. Subsequently, only the CU-OP lines, were followed for their responses to targeted inhibitor-chemotherapeutic drug combinations, and here also positive and neutral effects were disclosed.The data on the FDA approved drugs alpelisib and erdafitinib paralleled the inhibitory effects on viability and proliferation, as well as the effects on cytotoxicity and apoptosis of recently tested inhibitors BEZ235 and AZD4547 on UPCI-SCC-154 and UT-SCC-60A , 45. TheThat the cell lines above had drug dependent dose decreases in viability and proliferation, to FDA approved inhibitors, and that effects were enhanced upon combining the two was anticipated, since UPCI-SCC-154 and UT-SCC-60A had responded in an analogous way, to corresponding inhibitors BEZ235 and AZD4547  was superior to the included PI3K inhibitors with regard to inducing cytotoxicity , 42, 43.Furthermore, all TSCC/BOTSCC cell lines, with/without FGFR or PI3K mutations, were sensitive to PI3K and FGFR inhibitors and those with such mutations were not necessarily more sensitive or resistant to the inhibitors, similar to that reported for other tumor types , 46–53. The HPV+ and HPV- TSCC/BOTSCC cell lines were also examined for their responses to single cisplatin and docetaxel therapy and also here CU-OP-2 and -3 were the most resistant ones, while the remaining cell lines tended to be more sensitive. This pattern was not reflected upon by the effects of cisplatin and docetaxel on proliferation, which was largely inhibited in a similar way for all cell lines. At the doses used however, neither cisplatin or docetaxel disclosed prominent effects on cytotoxicity or apoptosis, but better effects may have been obtained if higher concentrations or repeated treatments had been used. Nonetheless, here we aimed to use as low doses as possible, in order to disclose combinational effects upon combining the chemotherapeutic agents with the inhibitors.When examining potential combinational effects using cisplatin and docetaxel together with BYL719 or JNJ-42756493, mainly neutral and positive, with occasional negative effects were disclosed. Of note were some positive combinations with BYL719 and cisplatin or docetaxel especially on CU-OP-2, but also with the other cell lines, and the data suggest it could be worthwhile elaborating further on optimizing the effects of such combinations.To our knowledge, the pursuit of potential positive effects of alpesilib and erdafitinib upon combination with cisplatin and docetaxel have not been examined before in TSCC/BOTSCC, and there are only limited studies on other cell lines, comprising some of the drug-inhibitor combinations in this report. One study on nasopharyngeal cancer cell lines, showed neutral or limited positive effects upon combining BYL719 and cisplatin, while another report on a non-small lung cancer cell, showed more positive effects , 54. HowThis report has limitations. Despite six cell lines were included, more could have been examined. Nonetheless, UPCI-SCC-154 and UT-SCC-60A have extensively been used and the CU-OP cell lines, have the benefit of including lines with PIK3CA and FGFR3 mutations –41. IrreTo conclude, this study supports the potential of further exploring the combined use of FDA approved drugs alpelisib and erdafitinib for the treatment of recurrent TSCC/BOTSCC especially when other options e.g., check point inhibitors are not useful.The original contributions presented in the study are included in the article/SH and OK did the majority of the experiments, interpreted the data, calculated the statistics and contributed to the writing of the manuscript. CB performed the CAST-PCR to detect the mutations of the cell lines presented in this study. NW and RU collaborated with SH and OK and performed some experiments contributed together with SH and OK in the graphs of the manuscript. NW and RU initiated the experiments and the interpretation of the initial experiments and contributed to the writing of the material and methods section, all under the supervision of SH. MZ and AN assisted in the analysis of the data and in their clinical interpretation. TD, SH, and OK made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data and have been involved in drafting the manuscript and revising it critically for important intellectual content. TD has also provided the sources of the performance of the experiments. TD, OK, SH, MZ, and RU provided the financial support for conducting the research project. All authors contributed to the article and approved the submitted version.This research was funded by the Swedish Cancer Foundation (grant no. 18 0440), the Stockholm Cancer Society (grant no. 181053), the Swedish Cancer and Allergy Foundation (grant no. 190), the Royal Swedish Academy of Sciences (grant no. 2017-2018), the Stockholm City Council (grant no. 20180037), Karolinska Institutet (grant no. 2018:0007) and Stiftelsen Clas Groschinsky Minnesfond , Lindhes Advokatbyrå , and University of Medicine and Pharmacy Grigore T Popa Iasi (grant no. 6983/21.04.2020).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Reproductive tract dysbiosis, due to the action of pathogens and/or unhealthy lifestyle, has been related to many reproductive diseases and disorders in mammalian species. Classically, such a problem has been confronted by the administration of antibiotics. Despite their effectiveness for controlling disease, treatments with antibiotics may negatively affect the fertility of males and females and, mainly, may induce antibiotic resistance. Accordingly, safer alternatives for maintaining reproductive system eubiosis, such as probiotics, are required. The present review summarizes the current knowledge on the biodiversity of the microbiota at the reproductive tract, possible changes in the case of dysbiosis, and their relationships with adequate reproductive health and functioning in both females and males. Afterwards, mechanisms of action and benefits of different probiotics are weighed since the biological activities of probiotics may provide a promising alternative to antibiotics for maintaining and restoring reproductive eubiosis and function. However, at present, it is still necessary for further research to focus on: (a) identifying mechanisms by which probiotics can affect reproductive processes; (b) the safety of probiotics to the host, specifically when consumed during sensitive reproductive windows such as pregnancy; and (c) the hazards instructions and regulatory rules required for marketing these biological-based therapies with sufficient safety. Thus, in this review, to draw a comprehensive overview with a relatively low number of clinical studies in this field, we showed the findings of studies performed either on human or animal models. This review strategy may help provide concrete facts on the eligible probiotic strains, probiotics colonization and transfer route, and prophylactic and/or therapeutic effects of different probiotic strains. Plenty of unfriendly factors can adversely affect the community of the reproductive tract’s microbiome in both human beings and/or animals because of either modern lifestyle or industrialization and intensification of production, leading to reproductive tract dysbiosis and the need for restoring a eubiotic state.pro that means “for” or “in support”, and the second clause is originated from the Greek adjective biotic that means “life”, so together, they mean “in support of life”. Scientifically, probiotics refer to viable microorganisms that have beneficial effects on the consumers’ health. Reproductive dysbiosis has been related to several reproductive disorders and infertility. The use of antibiotics and the use of probiotics are basically two hypothesized approaches that could be used to control reproductive tract microbiota. In this context, several studies have confirmed the beneficial effects of antibiotics to tackle many reproductive microbial infectious diseases and related infertility ,2,3. On Many studies have shown the positive effects of probiotics-based therapies on reproductive health in human ,7,8,9 anThe present review sheds the light on: 1- the relationships between reproductive tract eubiosis/dysbiosis and reproductive health and functioning; 2- the use of probiotics for maintaining and restoring reproductive eubiosis and function with emphasis on their properties, mechanisms of action, and benefits for reproductive function; 3- the safety of probiotics to the host; and 4- the litigation and regulatory rules required for marketing these biological-based therapies.Lactobacillus species, as well as Acinetobacter, Pseudomonas, Comamonadaceae, Jonquetella, Fusobacterium, Bacteroidetes, and Preovotella [Escherichia coli, Trueperella pyogenes, Enterococci, Enterobacteriaceae, Streptococci, Ureaplasma, and Staphylococci are classified as pathogenic microbes and have been linked to several reproductive disorders [A direct relationship between the biodiversity of reproductive tract microbiota and reproductive health and the completion of different reproductive events has been confirmed. The microbial biodiversity of the reproductive tract is highly dynamic and is affected by several internal and/or external factors. However, for maintaining adequate reproductive functioning and health, the prevalence of beneficial microbes should be ensured. The prevalence of some microbial species has been related to sufficient reproductive health and functions. Some microbial species are classified as beneficial microbial strains, mainly ovotella . In contisorders ,16,17.Either in males or females, reproductive tract dysbiosis has been related to many reproductive dysfunction and infertility. Dysbiosis (the dominance of pathogen-driven microbes) of the reproductive tract can disrupt numerous vital reproductive functions such as gametogenesis, steroidogenesis, embryo development and implantation, and establishment of pregnancy . Atopobium vaginae, Acinetobacter, Cloacibacterium, Comamonadaceae, Escherichia, Porphyromonas, and Pseudomonas [Bacteroides, Mobiluncus, Trichomonas vaginalis, and Candida species was seen in vaginal smears [Propionibacterium, Streptococcus, Actinomyces, Bifidobacterium, and Propionibacterium in their follicular fluid had less embryo transfer and pregnancy outcomes following in vitro fertilization and embryo transfer [Lactobacillus [In women, many gynecological diseases are related to the dysbiosis of endometrial microbiome, i.e., chronic endometriosis , dysfuncudomonas ,24. In wl smears . The dystransfer . In womebacillus ,28.In women, reproductive tract microbial biodiversity is highly dynamic and affected by several factors, mainly hormonal changes related to the stage of the reproductive cycle. High estrogen levels, such as around ovulation time, have been found to contribute to the maintenance of reproductive tract eubiosis. Estrogens maintain the acidity of the vaginal fluids, which makes them suitable media for the growth of beneficial bacteria lactobacilli. On the other hand, low levels of estrogens and the increment in pH such as that induced around menstrual period can shift the reproductive tract from eubiosis to dysbiosis through lowering the number of lactobacilli and increasing the opportunity of undesirable microorganisms’ growth, increasing the susceptibility of infectious pathogens ,30. Mycoplasma hominis was seen in infertile men than in fertile men [Neisseria, Pseudomonas, and Klebsiella accompanied by a reduction of Lactobacillus has recently been linked to oligoasthenoteratozoospermia and seminal hyper viscosity [Proteobacteria and a lesser population of the friendly bacteria Lactobacilli compared to those of healthy men [In men, the disbiosis of the male reproductive tract’s microbiome leads to several inflammatory diseases , subfertility, and poor semen quality . For exatile men . An incriscosity . In anotlthy men .Bacteroides and Enterobacteriaceae have been shown in cows with reproductive disease compared to healthy cows [Bacteroidetes and decreased Parvimonas, Porphyromonas, unclassified Veillonellaceae, and Mycoplasma was associated with bovine necrotic vulvovaginitis [Bacteroidetes and Fusobacteria was associated with metritis [Campylobacter in vaginal microbiota was associated with increased rates of abortion [The relationship between the reproductive tract microbiota biodiversity and reproductive health has also been reported in animals. The dominance of certain genera in the vaginal microbiota in female bovine have been linked to reproductive disorders. For instance, increased relative abundance of thy cows . In boviaginitis . The premetritis . In sheeabortion .These findings highlight the role of reproductive tract eubiosis in achieving adequate reproductive health and fertility and the importance of fixing any dysbiosis that might be raised due to unpleasant factors.The manipulation of the microbiota existing in the reproductive tract of both males and females can be an effective tool to control many reproductive diseases and associated infertility. Reproductive outcomes in both humans ,40 and aThe negative effects of antibiotics on reproductive organs and functions can be related to the lack of the specificity of broad-spectrum antibiotics, which could not only impair the growth of dysbiotic bacteria but also symbiotic bacteria. Furthermore, the widespread use of antibiotics poses a serious health risk due to the development of multiple-antibiotic-resistant microbial species. Furthermore, antibiotics-based treatments are linked to high rates of recurrent infections after treatment and side effects derived from the clearance of naturally harbored flora in other body sites . These aLactobacillus species , Bifidobacterium species, and Bacillus species are the major representatives of probiotics for modulating fertility dysbiosis [The term ‘probiotics’ has come to be used to refer to the live microorganisms that, when administered in adequate amounts, confer a health benefit to the host . More reysbiosis . The proProbiotic microbial species should have certain criteria to be approved as being safe compounds for health and effective when they are used for prophylactic and/or therapeutic interventions. In this term, safety properties  and functional properties  should be tested before approving any microorganism as a potential probiotic ,50. ThesLactobacillus rhamnosus GR1 and Lactobacillus Reuteri RC14 may persist up to 19 days in the human vagina following intravaginal administration. Both strains survive at low pH, have high adherent ability to uroepithelial and vaginal cells, and are able to colonize the vagina when administered orally, whereas some strains such as Lactobacillus rhamnosus GG and Lactobacillus acidophilus are not well-suited to colonize the vagina or to produce H2O2 , explaining why both these strains fail to prevent the recurrence of urogenital infections [Functional properties of probiotics are also important to achieve effective probiotics-based therapy. The properties such as tolerance to acids, bile salts, and lysozyme and gastrointestinal survival are related to the ability of these microorganisms to survive through the gastrointestinal system and to transfer to other target sites in body such as reproductive organs. Thus, these properties may be very important for oral-based administration to ensure the transfer of the probiotics to other sites of action, conferring local and direct effects on target organs. On the other hand, adhesion capacity may be more important when probiotics are vaginally administrated to allow a direct and targeted colonization action of the probiotics for restoring unhealthy vaginal microbiota ,62. In fLactobacilli isolated from the vaginas of healthy women for their potential safety and functional properties. Of 119 assessed strains, 7 identified strains  have been found to typically fulfill most criteria of probiotics. In the same study, 6 out of 119 strains belonging to Lactobacillus crispatus and Lactobacillus gassseri species have been found to exhibit phenotypical resistance to vancomycin. According to the previous facts on the eligibility criteria of microorganisms to act as an effective probiotic strain for maintaining and restoring reproductive health and functioning, it is supposed that one important potential source of probiotic strains is the reproductive tract itself . InnovatLactobacillus plantarum, fermentum and rhamnosus) with potential probiotic properties. These bacterial species also exhibit phenotypical resistance to vancomycin-resistant strains and sensitivity to streptomycin, erythromycin, kanamycin, and chloramphenicol [Enterococci may preclude this fear, as the antimicrobial resistance of these strains is attributed to the synthesis of a modified cell-wall peptidoglycan precursor and is different from the transferable mechanism observed for other bacteria [El-Deeb and co-workers  isolatedphenicol . In factbacteria .Lactobacillus rhamnosus BPL005 (CECT 8800) against the bacterial colonization of primary endometrial epithelial cells infected with Atopobium vaginae, Gardnerella vaginalis, Propioni bacterium acnes, and Streptococcus agalactiae pathogens. In the same context, Lactobacillus crispatus is the most frequent colonizing species in healthy women with antimicrobial activity, and it is therefore nominated to be used as a probiotic therapeutic alternative to antibiotics [Weissella confusa, Enterococcus hirae, Leuconostoc lactis, and Ilyobacter polytropus. However, these strains showed different hydrophobicity, antibacterial activity, and adherence ability. Thus, the authors suggested that the use of a mixture of these strains can be a more efficient intervention for developing probiotic products that can prevent and treat reproductive tract infections in cows. Similarly, Ametaj and co-workers [Lactobacillus sakei FUA 3089, Pediococcus acidilactici FUA 3138, and FUA 3140, isolated from the vaginal tract of healthy cows to decrease the incidence of metritis, pyometra, and vaginal purulent discharges, as well as the number of cows with abnormal cervical size and uterine fluctuation. In addition, vaginal isolates of dromedary camels (Camelus dromedarius) have been found to contain Lactobacillus species belonging to Lactobacillus plantarum, Lactobacillus fermentum, and Lactobacillus rhamnosus strains with potential probiotic properties. These bacterial species showed strong antimicrobial activity against Staphylococcus aureus, which is considered as one of the most common bacterial causes of endometritis in dromedary camels and other animal species [The probiotic properties of microbial species isolated from different sites of the reproductive tract has been confirmed in many studies. Chenoll and co-workers  confirmeibiotics . In anot-workers  proved t species . Overall, these studies performed either on human or animal models confirm that the reproductive tract microbial communities represent a suitable source of probiotic candidates that could be used in new functional formulates for maintaining and restoring reproductive tract eubiosis.Lactobacilli drop, whilst there is an overgrowth of other groups such as Gardnerella, Atopobium, Prevotella, and Streptococcus, among others [Lactobacilli, in the uterine lumen and vaginal microbiome help to maintain acidic milieu, which provides an efficient protective agent against pathogenic microorganisms [Probiotics can maintain and restore reproductive tract eubiosis through different mechanisms. Probiotics have been found to produce bioactive molecules that have direct effects on reproductive tract microbiota, as these bioactive molecules can act as antimicrobial agents. In a pathogenic process, the microbiome shifts to a dysbiotic state, in which pH is increased to values above 4.5 and g others . The prorganisms .Some probiotic species, in addition to the ability of many species to reduce the pH, are able to produce oxidative molecules such as hydrogen peroxide, as well as another group of molecules known as bacteriocins . 2+ or Cu2+; (2) the synthesis of their own antioxidant enzymes such as Fe- or Mn-superoxide dismutase and Catalase; (3) the production of metabolites with antioxidant activity, such as glutathione, butyrate, and folate; (4) controlling antioxidant signaling pathways ; and (5) the regulation of the enzymes producing reactive oxygen species such as NADPH oxidase complex [As shown in  complex .adrenal (HPA) axis. Some probiotic species such as Bifidobacterium infantis and Lactobacillus rhamnosus (JB-1) and farciminis have been found to exhibit anxiolytic effects and improve psychiatric-disorder-related behaviors, including anxiety, depression, acute stress disorders, and obsessive disorders [Other studies have highlighted another mechanism of action of probiotic that is mediated by the hypothalamic–pituitary–isorders ,72. Thesisorders .vaginal odor, burning with urination, and itching. Additionally, it is linked to the increased risk of early delivery (pre-term delivery) among pregnant women [Lactobacillus vaginal tablets revealed the disappearance of bacterial vaginosis symptoms and had a clinically normal vaginal microbiota after daily probiotic therapy application for just 7 days. Furthermore, long-term vaginal administration of Lactobacillus rhamnosus appears to be a useful, safe, and effective antibiotic complementary approach in the management of bacterial vaginosis [Lactobacillus rhamnosus GR1 and reuteri RC14 in pregnant women restores the normal vaginal flora and acidic pH and interrupts the infectious/inflammatory process due to bacterial vaginosis with recovery rates comparable to those of tocolytic therapies. Thus, probiotics are preferred over tocolytic therapy to reduce the adverse maternal and fetal outcomes [Bifidobacterium lactis V9 was an effective treatment for tackling polycystic ovary syndrome, a hormonal-disturbance-induced disease. As hypothesized by the authors of this study, the effect of Bifidobacterium lactis V9 is mediated by the gut–brain axis, as Bifidobacterium lactis V9 produces certain amounts of short-chain fatty acids, which consequently increase the release of ghrelin and peptide YY. These latter two molecules can restore the hormonal profile of sex hormones, mainly LH and prolactin, at the brain level by a manner similar to normal hormonal profiles.Among reproductive diseases in women, bacterial vaginosis represents one of the major pathogenic diseases in women of reproductive age. Bacterial vaginosis is caused by vaginal dysbiosis and predominance of pathogenic microorganisms . It is characterized by several clinical and uncomfortable symptoms such as the release of discharge with abnormal nt women . In manynt women ,13; in taginosis . In addioutcomes . Probiotoutcomes  found thLactobacillus rhamnosus HN001 (6 × 109 CFU/day) to women during early pregnancy and during the first 6 months of breastfeeding can reduce the rates of infant eczema and atopic sensitization at 1-year and maternal gestational diabetes mellitus, bacterial vaginosis, and depression and anxiety postpartum [Interestingly, probiotics can be safely used during pregnancy and breastfeeding to offer a unique opportunity to influence a range of important maternal and neonatal outcomes. The oral supplementation with the probiotic stpartum . The finThe positive effects of probiotics on female reproduction are not only observed in humans but also in animal models. Specifically, farm animals may be of particular importance, as maintaining their health, improving reproductive and productive performance, and eliminating drugs/antibiotics use represent crucial challenges. Considering these aspects, many probiotic microbial species have been tested either for treating reproductive diseases or for improving the productivity of healthy farm animals .Lactobacillus species  at the time of the insertion of fluorogestone acetate (FGA) sponges for estrus synchronization in ewes restored normal vaginal microflora and improved fertility . For example, the management of the estrous cycle in some farm animals  includes many protocols that depend on intravaginal progesterone devices and/or sponges. In many cases, the administration of these intravaginal means has been found to generate histological and cytological alterations  at the time of its withdrawal, which evoke inflammatory responses and provoke abnormal vaginal discharges, impacting animal welfare aspects and subsequent reproductive performance . In a reLactobacillus sakei FUA3089, Pediococcus acidilactici FUA3138, and Pediococcus acidilactici FUA3140 had smaller cross-sectional areas of gravid horn and uterine body on day 14 postpartum, earlier uterine involution, and resumed ovarian cyclicity earlier. Generally, the positive effects of probiotic treatments on reproductive performance and cows’ health might be ascribed to the ability of Lactobacillus species to decrease postpartum uterine infection and thus to decrease endotoxin translocation into the systemic circulation, decreasing the occurrence of inflammatory responses [In addition, probiotics-based treatments can be used to improve the postpartum health and reproductive performance of farm animals. Specifically, parturition and uterine involution during the postpartum period may expose females to many reproductive infectious diseases, mainly due to the associated disturbance in the metabolism and the increase in inflammatory responses ,77,78,81Clostridium butyricum  from day 90 of gestation to weaning at Day 21 postpartum could shorten the duration of farrowing and enhance the growth performance of suckling piglets. Moreover, 0.2% Clostridium butyricum administration to sows changed the composition of intestinal microbiota, specifically increasing the relative abundance of Prevotella [Bacillus species serve as safe and effective feed additives which can improve the reproductive performance of farm animals [Bacillus subtilis and/or Bacillus licheniformis improved the lipid and protein metabolism profiles of sows, the placental antioxidant capacity, and placental efficiency that resulted in greater piglet birth weight. In sheep, the administration of monensin or Saccharomyces cerevisiae improved the metabolic status and ovarian steroid production of dams, as well as the growth performance of lambs, highlighting the possibility of using probiotic Saccharomyces cerevisiae as an alternative to the antibiotic monensin [Focusing on improving the productivity of animals, in sows, the addition of dietary evotella . In addi animals ,83. Gu a animals  found thmonensin . The finA summary of some studies on the effects of probiotics on men’s reproductive health and fertility is shown in These positive effects have been seen in different infertility-related models. Infertility related to age , obesity9 CFU/mL of Lactobacillus acidophilus, Bifidobacterium spp., Bacillus coagulans, and Lactobacillus rhamnosus), for 63 consecutive days, reversed all negative effects of NAFLD on semen quality and redox homeostasis in rats. Other studies  [The candidate probiotic species, in addition to health safety as discussed above, should have functional and industrial properties, enabling them to be stable during pharmaceutical processing and storage and to survive afterwards in different biological environments, such as the gastrointestinal and reproductive tracts. In this context, the emergence of some recent industrial technologies such as micro/nanoencapsulation technology can help to improve the stability of probiotics . The emecterium)  and mushcterium)  in the fCurrently, the most recent research trends refer to the possibility of the use of postbiotics  as an alternative to probiotics itself. The use of postbiotics might have offered several advantages over probiotics, such as obtaining a specific mechanism of action and biological role, an easier production process for industrial-scale-up, and, lastly, a better availability and accessibility during marketing and application .Lactobacillus, Bifidobacterium, and Bacillus are the most adopted for modulating reproductive tract dysbiosis [The dysbiosis of the reproductive tract microbiota due to the action of pathogens and/or unhealthy lifestyle has been related to many reproductive diseases and disorders in both humans and animal models. Recent studies have focused on the possible roles of different probiotics in restoring the reproductive tract eubiosis. Among the different candidate species, ysbiosis . The bioysbiosis . A highlysbiosis ,52. Hence, future studies have to focus on identifying mechanisms by which probiotics can affect reproductive processes, the safety of probiotics to the host specifically when they are consumed during sensitive reproductive windows such as pregnancy, and finally, the litigation and regulatory rules required for marketing these biological-based therapies with sufficient safety and hazards instructions .


The clinical translation of mesenchymal stem cells (MSCs) is limited by population heterogeneity and inconsistent responses to engineered signals. Specifically, the extent in which MSCs respond to mechanical cues varies significantly across MSC lines. Although induced pluripotent stem cells (iPSCs) have recently emerged as a novel cell source for creating highly homogeneous MSC (iMSC) lines, cellular mechanosensing of iMSCs on engineered materials with defined mechanics is not well understood. Here, we tested the mechanosensing properties of three human iMSC lines derived from iPSCs generated using a fully automated platform. Stiffness-driven changes in morphology were comparable between MSCs and iMSCs cultured atop hydrogels of different stiffness. However, contrary to tissue derived MSCs, no significant changes in iMSC morphology were observed between iMSC lines atop different stiffness hydrogels, demonstrating a consistent response to mechanical signals. Further, stiffness-driven changes in mechanosensitive biomarkers were more pronounced in iMSCs than MSCs, which shows that iMSCs are more adaptive and responsive to mechanical cues than MSCs. This study reports that iMSCs are a promising stem cell source for basic and applied research due to their homogeneity and high sensitivity to engineered mechanical signals. MSCs are present in numerous stem cell niches including bone marrow and adipose tissue and can be expanded in vitro by plating onto tissue culture polystyrene (TCPS), which causes them to adhere, adopt a spindle-like shape, and proliferate into fibroblastic colony-forming units2. Owing to their unique properties and ease of expansion, MSCs have been extensively studied and used in numerous clinical trials for the treatment of various medical disorders4.Mesenchymal stem cells (MSCs) are non-hematopoietic cells capable of differentiating into cells that produce various mesodermal tissues, including osteoblasts, adipocytes, and chondrocytes5, which influence numerous functions including migration, differentiation, and paracrine signaling6. To regulate stem cell behavior outside of the body, biomaterials are used to recapitulate specific properties of tissue microenvironments. For example, ECM elasticity and tissue-level stiffness are strong drivers of cellular mechanosensing and phenotypic commitment in vitro7. Engler et al. showed that MSCs atop soft hydrogels that mimic the stiffness of brain tissue express neuronal biomarkers, whereas MSCs on rigid substrates produce osteocalcin, a bone tissue-specific protein secreted by osteoblasts7. On a molecular level, MSC mechanosensing is led by several mechano-transducer proteins that collectively induce changes in focal adhesion maturation9, cytoskeletal contractility/alignment11, and nuclear Yes-associated protein (YAP) localization12.In vivo, the multifunctional phenotype of MSCs is regulated by chemical and physical cues of the tissue microenvironment13, and donor variability between MSC lines derived from adult tissues is significant, resulting in inconsistent responses to engineered signals14. These challenges limit the possibility of manufacturing high quality, homogeneous MSC lines in large numbers needed for basic research and stem cell-based therapies15. When successfully reprogrammed, induced pluripotent stem cells (iPSCs) reset any possible mechanical memory that could bias their response to engineered materials by erasing historical epigenetic, transcriptional, and non-genetic information17. Indeed, iPSCs have recently been differentiated into functional MSCs (iMSCs), displaying phenotypic similarities with tissue derived MSCs19. However, there are only a limited number of studies that have explored the use of iMSCs20, and the effects of mechanical signals on iMSC mechanosensing is not well understood. In this study we sought to test two hypotheses: (1) iMSCs “feel” mechanical properties resulting in changes in morphology and intracellular mechanosensitive protein organization  comparable to MSCs, and (2) iMSC mechanosensitivity to substrate stiffness is more reproducible within iMSC populations and across iMSC lines as a consequence of the reprogramming process, which erases mechanical memories acquired during development and in response to engineered biophysical cues.Although engineered mechanical cues can regulate MSC mechanosensing in vitro, MSC populations are heterogeneous21. We report that iMSCs are more responsive to matrix stiffness than human MSCs derived from adult tissues, and that stiffness-mediated changes in cellular mechanosensing are more consistent across different iMSC lines.To test our hypotheses, we evaluated cell-material interactions of three human iMSC lines derived from iPSCs generated using a robotic, fully automated platform, which results in the production of highly reproducible iMSC lines21.Manual production of human iPSC lines is time consuming and can result in significant line-to-line variability. To enable consistent production of high-quality and highly reproducible iMSC lines, fibroblasts from human skin biopsies were reprogrammed into stem cells using the NYSCF Global Stem Cell Array®, a modular, robotic platform for high-throughput production, maintenance, and differentiation of iPSCs Fig. a. Three 23. Interestingly, in vitro expansion over ten passages also results in a reproducible and progressive increase in average cellular length, which is a phenomenon also observed in tissue derived MSCs  class II, which suggests iMSCs are more immunoprivileged than adult MSCs26. Notably, iMSCs express more integrin alpha 2 (CD49b), integrin alpha 3 (CD49c), and integrin alpha 4 (CD49d) than adult MSCs , intermediate (10 kPa), and stiff (20 kPa) HA hydrogels30. Thus, we sought to synthesize HA hydrogels with a stiffness range of 5–20 kPa to study the effects of mechanics on iMSC shape and mechanotransduction.While there are many macromers that can be used to synthesize hydrogels, hyaluronic acid (HA) hydrogels are highly biocompatible and amenable to extensive biochemical and biophysical modifications32. The amount of macromer (3 wt%) was kept constant and Low (5.19 ± 1.04 kPa), Med (9.58 ± 0.98 kPa), and High (19.27 ± 2.41 kPa) matrix stiffness hydrogels were formed by varying the amount of crosslinker added  macromers with thiols in DTT crosslinkers as previously reported2, respectively  is known to initialize at least two signaling pathways of MSC mechanosensing and plays an important role in controlling several cellular processes including cell spreading, migration, and focal adhesion maturation37. After three days in culture, all iMSC lines displayed an increase in focal adhesion maturation with increasing matrix stiffness. pFAK length for iMSCs on Low, Med, and High stiffness hydrogels was 3.94 ± 0.56, 5.98 ± 0.62, and 7.18 ± 0.56 µm, respectively  on Med stiffness hydrogels. Differences between iMSC and MSC morphology were not as pronounced on Low or High stiffness hydrogels  stiffness substrates, representative cell silhouettes of iMSCs Fig. a, top quHistograms of cell area show a homogeneous distribution for iMSCs Fig. b, top. INext, we examined scatter plots of nuclear YAP versus area of iMSC Fig. d, top anActin anisotropy values for iMSCs were also consistent across different lines while for MSCs actin anisotropy was significantly different across cell lines Fig. f. These 39. Due to their remarkable sensitivity and homogeneity, iMSCs could be a viable source for large scale manufacturing of human stem cells for both autogenic and allogeneic cell therapies. As we continue to increase our understanding of iMSC-material interactions, we also believe that iMSCs will emerge as a new class of cells for regenerative medicine and tissue engineering applications.In this study, we derive iMSCs from iPSCs and demonstrate that iMSCs are more homogeneous and mechanosensitive than MSCs isolated from adult tissues. This finding resulted from evaluating iMSC morphology and matrix mechanosensing on mechanically defined 2D hydrogels and motivates future studies that investigate iMSC-material interactions in more complex and physiologically relevant environments. To this end, the thiol-norbornene chemistry used here can be easily adapted to form hydrogels that support 3D cell culture and spatial patterning of biophysical and biochemical signals21. All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by The New York Stem Cell Foundation Research Institute. Skin biopsies were shared as deidentified following written informed consent. Generated iPSC lines were characterized via confirmation of pluripotency markers OCT4 and TRA-1-60 using immunostaining and fluorescence imaging. Additional characterization was performed via global surface marker profiling using a BD Lyoplate Human Cell Surface Marker Screening Panel (BD Biosciences) per manufacturer’s instructions. Using chemically defined differentiation medium, iPSC lines were differentiated towards osteoblasts, chondrocytes, or adipocytes and evaluated using von Kossa , Alcian blue , or Oil red O (intracellular triglycerides), respectively.Fibroblasts from skin biopsies were programmed into iPSCs using the Global Stem Cell Array® as previously reported1H NMR spectra used to calculate percent of HA repeat units functionalized with Nor- and Me- is in Supplementary Fig. Sodium hyaluronate (NaHA) was first converted to its tetrabutylammonium salt (HA-TBA). To synthesize HANor, the carboxylic acid residues of HA-TBA were modified with 5-norbornene-2-methylamine . To synthesize HANorMe, the hydroxyl residues of HANor were modified with methacrylic anhydride . To biofunctionalize HANorMe with RGD, a Michael addition reaction between thiolated RGD (cRGD) peptide and methacrylates was performed . Representative 2). Individual hydrogels were removed from the molds and placed in 1 mL of PBS to swell overnight at 37 °C before mechanical testing. Compressive moduli were determined using a Shimadzu EZ-SX Mechanical Tester running at a constant strain rate of 10%/min. The modulus was calculated from the slope of the stress–strain curve between 10 and 20% strain (n ≥ 3).HANor was dissolved in phosphate buffer saline (PBS) at 3 wt% with varying amounts of DTT and 0.05 wt% I2959. The prepolymer solution (80 µL) was pipetted into a silicone mold  and irradiated with UV light .Stem cell lines from the New York Stem Cell Foundation Research Institute expanded to passage 4 in growth media  were used in all experiments. Human mesenchymal stem cell lines (Lonza) were expanded to passage 4 using the culture media described above. Cells were then cultured on top of Low, Medium, or High stiffness hydrogels , phalloidin (actin), and/or Hoescht (nuclei). Samples were then imaged using a Nikon A1 confocal microscope and ImageJ software was used to calculate morphology  and cellular mechanosensing  parameters.All data are from three independent biological experiments. At least 50 cells per treatment and biological experiment were quantified. For three group comparisons, one way ANOVA between groups (α = 0.05) was performed using GraphPad Prism. If the results of the ANOVA were found to be significant, post hoc analysis was performed using the Tukey multiple comparisons test to compare results among groups. Hierarchical clustering of surface marker data was generated using the analysis software Morpheus (Broad Institute) based on Euclidean distance.Supplementary Information.