The mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) signaling controls cell metabolism, promotes cell survival, and contributes to tumorigenesis, yet its upstream regulation remains poorly defined. Although considerable evidence supports the prevailing view that amino acids activate mTOR complex 1 but not mTORC2, several studies reported paradoxical activation of mTORC2 signaling by amino acids. We noted that after amino acid starvation of cells in culture, addition of an amino acid solution increased mTORC2 signaling. Interestingly, we found the pH of the amino acid solution to be alkaline, ∼pH 10. These observations led us to discover and demonstrate here that alkaline intracellular pH (pHi) represents a previously unknown activator of mTORC2. Using a fluorescent pH-sensitive dye (cSNARF1-AM) coupled with live-cell imaging, we demonstrate that culturing cells in media at an alkaline pH induces a rapid rise in the pHi, which increases mTORC2 catalytic activity and downstream signaling to the pro-growth and pro-survival kinase Akt. Alkaline pHi also activates AMPK, a canonical sensor of energetic stress. Functionally, alkaline pHi activates AMPK-mTOR signaling, which attenuates apoptosis caused by growth factor withdrawal. Collectively, these findings reveal that alkaline pHi increases mTORC2- and AMPK-mediated signaling to promote cell survival during conditions of growth factor limitation, analogous to the demonstrated ability of energetic stress to activate AMPK–mTORC2 and promote cell survival. As an elevated pHi represents an underappreciated hallmark of cancer cells, we propose that the alkaline pHi stress sensing by AMPK–mTORC2 may contribute to tumorigenesis by enabling cancer cells at the core of a growing tumor to evade apoptosis and survive. These mTORCs sense and integrate diverse extracellular and intracellular signals derived from hormones, growth factors, nutrients, and energy through distinct downstream substrates to control cell physiology in ways appropriate for biological context , 4, 5. mskeleton , 4, 5. NActivation of mTORC1 by the cooperative action of insulin and amino acids has been studied extensively. In response to insulin, activation of the PI3K-Akt-tuberous sclerosis complex (TSC) pathway leads to Ras homolog enriched in brain (Rheb)-GTP–mediated activation of mTORC1 on the surface of lysosomes in a manner that requires sufficient levels of amino acids , 6, 7, 8i.e., containing amino acids) activated mTORC2 as well as AMPK. Interestingly, we measured the pH of the amino acid solution and found it to be an alkaline ∼pH 10. When we adjusted the pH of the amino acid solution to physiological pH 7.4, it failed to increase mTORC2 or AMPK signaling. These key observations enabled us to discover and demonstrate here that culturing cells in media at an alkaline pH activates the AMPK–mTORC2 axis through an increased intracellular pH (pHi), which attenuates apoptosis caused by growth factor withdrawal. As an elevated pHi represents an underappreciated hallmark of cancer cells . These results are consistent with a limited number of prior reports . Curiously, the amino acid solution increased phosphorylation of AMPKα on its activation loop site (S172) as well as the phosphorylation of mTOR S1261 and ACC S79, direct substrates of AMPK  . On their face, these data agree with prior reports that amino acids increase mTORC2 signaling , or DMEM lacking amino acids is used to deprive cells of amino acids, whereas addition of an amino acid solution containing total  amino acids, or re-feeding of cells with complete media, is used to stimulate cells with amino acids. While studying the mTORC1 response to amino acids, we noted that after starving cells of amino acids in D-PBS (which contains glucose) with or without dialyzed fetal bovine serum (FBS) (dFBS), addition of a commercial amino acid solution increased mTORC2 signaling, as measured by the sensitivity of Akt S473 phosphorylation to the mTOR inhibitor Torin 1  , 43. As ignaling , 35, 36 ignaling .Figure 1i.e., P-Akt-S473 and -T308) and AMPK  signaling regardless of amino acid status in both the presence and absence of serum growth factors . As expected, DMEM containing but not lacking amino acids increased mTORC1 signaling  regardless of the pH . Upon adjusting the pH of the amino acid solution, its addition at pH 10 but not 7.4 to cells in D-PBS (+dFBS) increased mTORC2 and AMPK signaling . These results indicate that the alkaline pH of the amino acid solution, not the amino acids per se, increased mTORC2 and AMPK signaling.When we used DMEM lacking amino acids rather than D-PBS to amino acid deprive cells, however, addition of the amino acid solution induced a rapid change in DMEM color from orange-red to purple, indicating a change from physiological pH ∼7.4 to alkaline pH. Thus, we measured the pH of the amino acid solution and found it to be an alkaline pH ∼10. To compare how alkaline pH and amino acids control mTORC2 signaling, we adjusted the pH of DMEM lacking or containing amino acids to 7.4 or 8.3. DMEM at pH 8.3 but not 7.4 increased mTORC2 (A (right) replicates the results of Tato et al.  or DMEM  failed to increase mTORC2 signaling. Similar to our results, Dalle Pezze et al. . These effects were rapid and transient, with maximal phosphorylation of Akt occurring at 5 to 15 min with apparent declines by 30 to 60 min. The activating effect of the alkaline extracellular pH on Akt phosphorylation was Torin 1 sensitive in all three cell lines (B and B and C), thus confirming increased mTORC2 signaling. In MEFs, pH 8.3 to 8.5 mediated maximal mTORC2 signaling (D). These results demonstrate that the alkaline extracellular pH increases mTORC2 signaling, presumably by increasing the pHi. It is important to note that shifting cells to media at alkaline pH has been shown to increase the pHi rapidly (4Cl). Addition of NH4Cl to cell culture media increases the cytosolic pH transiently upon rapid diffusion of NH3 (a weak base) into the cell and conversion to ammonium ion, NH4+, by scavenging protons from the cytosol, thus acidifying the cytosol .We next tested whether culturing cells in media at an alkaline pH is sufficient to increase mTORC2 signaling in the absence of changes in amino acid levels. Re-feeding mouse embryonic fibroblasts (MEFs), HEK293T cells, and U2OS cells with complete DMEM (containing amino acids and FBS) at pH 8.3 but not pH 7.4 increased Akt phosphorylation (P-S473 and P-T308) Fig. 2A and A–Ces B and , B and Cignaling D. These  rapidly , 45. To  cytosol , 47. Sloysosomes . We foun+–Na+ antiporter on the plasma membrane that drives proton (H+) efflux from the cytosol to the extracellular space. Thus, cariporide decreases the pHi . Taken together, these results demonstrate that the increased pHi mediates the effect of the alkaline extracellular pH on mTORC2 signaling.To confirm that culturing cells in media at alkaline pH activates mTORC2 by increasing the pHi, we used the drug cariporide. Cariporide inhibits NHE1, a H the pHi , 49, 50.in vitro kinase assays. To do so, we refed MEFs with DMEM, pH 8.3, immunopurified mTORC2 by immunoprecipitation of Rictor (a partner protein exclusive to mTORC2), and measured phosphorylation of His-Akt1 by mTORC2 in vitro. Indeed, the alkaline extracellular pH increased mTORC2 catalytic activity robustly, presumably by increasing the pHi (E). To demonstrate unambiguously that the increased pHi increases Akt S473 phosphorylation via mTORC2, we used Rictor−/− MEFs. Indeed, refeeding with DMEM, pH 8.3, increased P-Akt-S473 in Rictor−/− MEFs rescued with HA-Rictor but not vector control (V) (F). Taken together, these results show that a more alkaline pHi activates mTORC2 to increase downstream signaling to Akt.To test whether alkaline extracellular pH increases mTORC2 intrinsic catalytic activity, we performed mTORC2  the pHi E. To demtrol (V) F. Taken A). To confirm that cariporide attenuates the increase in the pHi caused by shifting cells to media at alkaline pH, we pretreated cells with cariporide before the shift to DMEM, pH 8.3, and monitored the pHi using cSNARF1-AM and live-cell imaging. It is important to note that the NHE family consists of several family members, and cariporide inhibits only a subset with differing potencies; in addition, cells express other proton (H+) transporters . In addition, we confirmed that addition of NH4Cl to DMEM at physiological pH 7.4 induces a rapid but transient rise in the pHi, as expected . These results confirm that shifting cells to media at alkaline pH or addition of NH4Cl to media at the physiological pH leads to a rapid increase in the pHi, consistent with other studies  that is capable of measuring changes in the pHi between 7 and 8. cSNARF1-AM undergoes a pH-sensitive shift in fluorescence wavelength emission depending on the protonation state. With 488-nm excitation, peak emission occurs at 580 nm in the protonated state (more acidic) and 640 nm in the deprotonated state . Thus, ratiometric imaging (640:580 nm) enables detection of changes in the pHi within the physiological range of normal cells (∼pH 7.2) and cancer cells (∼pH 7.4–7.6) , 51. MEFFig. 3sporters . Thus, c studies , 45.Figue.g., Rictor) . When MEFs incubated in D-PBS/dFBS were stimulated with the alkaline amino acid solution or refed with D-PBS/dFBS adjusted to pH 9.5 , we found that alkaline extracellular pH increased mTORC2 signaling in a manner partly dependent on AMPK (A and B). As expected, AMPK DKO abrogated AMPK signaling  (A and B). Alkaline extracellular pH was also sufficient to increase mTORC2 signaling in a manner partly dependent on AMPK in cells cultured in complete media  (C). From these results, we conclude that alkaline pHi increases mTORC2 signaling through AMPK and another unknown signal(s).Our prior work demonstrated that energetic stress increases mTORC2 catalytic activity and signaling directly through AMPK-mediated phosphorylation of mTOR and mTORC2 partner proteins ( Rictor) . As the  on AMPK , A and Bor-S792) , A and BMEM/FBS) C. From t2+ stores and raise cytosolic Ca2+  to activate AMPK depending on context . As expected, STO-609 reduced the ability of DMEM, pH 8.3, to activate AMPK, as shown by reduced P-AMPK-S172 and P-mTOR-S1261 (A). These results suggest that Ca2+ and CaMKKβ are required for activation of AMPK and mTORC2 signaling in response to the increased pHi.We next investigated how alkaline pHi activates AMPK. As the increased pHi has been reported to mobilize Calic Ca2+ , 53, and context , 54. We g to Akt , A and BOR-S1261 A. These i.e., with FBS) or serum-free DMEM  for an additional 3 h. As expected, cells maintained in serum-free media for the full 19 h displayed increased apoptosis relative to those rescued with complete media for the last 3 h, as monitored by blotting for cleaved caspase 3 and cleaved Parp (D). Consistent with our hypothesis that alkaline pHi protects against apoptosis, refeeding MEFs with serum-free DMEM at alkaline pH 7.8  suppressed apoptosis (D). Importantly, Torin 1 blocked the ability of DMEM at alkaline pH to suppress apoptosis, indicating a requirement for mTOR activity. While the prolonged alkaline extracellular pH  could not maintain Akt S473 phosphorylation, it maintained inhibitory phosphorylation of the Akt substrate and proapoptotic protein BAD (P-S136) (D). Consistent with our hypothesis that AMPK is required for alkaline pHi to protect against apoptosis, DKO of AMPKα1/α2 partially suppressed the ability of serum-free DMEM at alkaline pH 7.8 to attenuate apoptosis caused by growth factor withdrawal  D. ConsisF). It is important to note that a recent study also found that the alkaline pHi increases Akt P-S473, although mechanistic details were not defined (B-right), suggesting that another unknown signal responds to the alkaline pHi to increase mTORC1 and perhaps mTORC2 signaling (F). We speculate that prior studies reporting activation of mTORC2 signaling by amino acids . We thus propose that alkaline pHi increases cytosolic Ca2+ and activates CaMKKβ, which results in CaMKKβ-mediated activation of AMPK , delivered to cells as either NH4Cl or NH4OH, activates mTORC2 signaling in a manner that requires Ca2+ mobilization. Although they did not determine the mechanism by which NH4Cl or NH4Cl-induced Ca2+ mobilization increases mTORC2 signaling, they speculated involvement of a pH-mediated effect. Our results may explain their results, at least in part.Our findings suggest a mechanism for how alkaline pHi activates AMPK–mTORC2 signaling. CaMKKβ inhibition or Caling see , A and B of AMPK , 43 and  of AMPK . In supp+ stores , 53. Add  flux   and production of metabolic acids, many cancer cells in fact possess a slightly basic cytosol  include p53, EGF receptor, Ras-GRP1, and β-catenin  , 50, 51. cytosol , 38, 51. cytosol , 51. Doc-catenin , 58, 59.tastasis . Indeed,la model .By identifying the alkaline pHi as an activator of AMPK–mTORC2 signaling that attenuates apoptosis caused by growth factor withdrawal, our work suggests that the AMPK–mTORC2 axis may drive tumorigenesis in response to dysregulated pH dynamics by enabling growth factor–deprived, nutrient-deprived, and oxygen-deprived cancer cells at the core of a growing tumor to survive. Such a role is consistent with the paradoxical role of AMPK as a tumor promoter in certain contexts (despite its established role as a tumor suppressor) and the newfound role for AMPK–mTORC2 signaling in cell survival during energetic stress , 65, 66.2. Rictor−/− MEFs rescued with vector control or HA-Rictor were generated as described , the NaHCO3 concentration was increased from 44 mM to 120 mM by directly dissolving in NaHCO3 followed by filter sterilization and equilibration overnight in an incubator at 7.5% CO2, as described  were cultured in DMEM containing high glucose [4.5 g/l], glutamine [584 mg/l], and sodium pyruvate [110 mg/l] (Life Technologies #11995-065) supplemented with 10% FBS (Life Technologies #10347-028) and incubated at 37 °C in a humidified atmosphere with 7.5% COescribed . To effeescribed . As an a2. Images were acquired using a 40× oil objective (1.3 NA) at a resolution of 1024 × 1024 and an optical thickness of 1.18 μm . cSNARF1-AM signal was excited with an argon laser at 488 nm, and images sets were collected simultaneously in two emission band-pass filters at 553 to 618 nm (for 580 nm) and 663 to 738 nm (for 640 nm). Nikon NIS-Elements software was used to create and pseudocolor the 640:580 nm ratiometric images with values ranging from 0 (violet) to 2.0 (red). MetaMorph software was used to quantify and graph the ratiometric images by measuring the average ratio of a region of interest within one cell. Twenty five cells per field times four fields were quantified (n = 100 cells). A change in the 640:580 nm ratio and accompanying pseudocolored image reflects a change in the pHi.MEFs were plated in 8-well cover glass bottom chambers (Lab-tek #155409) and loaded with cSNARF-1-AM [10 μM] (Thermo Fisher #C1272), a fluorescent pH-sensitive dye, for 30 min at 37 °C in serum-free DMEM, which facilitates cSNARF1-AM loading. The cells were then rinsed with serum-free DMEM and refed with DMEM/FBS [10%], pH 7.4. Live-cell imaging (0–10 min) of cells cultured in DMEM/FBS, pH 7.4 or 8.3, was performed using a Nikon A1 confocal microscope equipped with a stage-top incubator that maintains the temperature and COAll data are contained in this article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Pancreatic cancer is a devastating disease with the worst survival rates of all cancers. Advances in immunotherapy have revolutionized the treatment of cancer, providing cures to patients with a previously lethal disease. Sadly, clinical trials of currently established immunotherapies have not provided significant therapeutic benefits to pancreatic cancer patients. For this reason, alternative treatment approaches that could sensitise the pancreatic tumours to immunotherapy are needed. Therapeutic focused ultrasound is a non-invasive physical modality that can kill cells by raising the temperature, and by disrupting and destroying tissue mechanically. It can also sensitise tumours to the effects of radiotherapy. This review article describes the use of therapeutic focused ultrasound in the treatment of pancreatic cancer. Attention is given to the immunological effects induced by therapeutic focused ultrasound, and how these can be used to sensitise the pancreatic tumours to immunotherapy.Traditional oncological interventions have failed to improve survival for pancreatic cancer patients significantly. Novel treatment modalities able to release cancer-specific antigens, render immunologically “cold” pancreatic tumours “hot” and disrupt or reprogram the pancreatic tumour microenvironment are thus urgently needed. Therapeutic focused ultrasound exerts thermal and mechanical effects on tissue, killing cancer cells and inducing an anti-cancer immune response. The most important advances in therapeutic focused ultrasound use for initiation and augmentation of the cancer immunity cycle against pancreatic cancer are described. We provide a comprehensive review of the use of therapeutic focused ultrasound for the treatment of pancreatic cancer patients and describe recent studies that have shown an ultrasound-induced anti-cancer immune response in several tumour models. Published studies that have investigated the immunological effects of therapeutic focused ultrasound in pancreatic cancer are described. This article shows that therapeutic focused ultrasound has been deemed to be a safe technique for treating pancreatic cancer patients, providing pain relief and improving survival rates in pancreatic cancer patients. Promotion of an immune response in the clinic and sensitisation of tumours to the effects of immunotherapy in preclinical models of pancreatic cancer is shown, making it a promising candidate for use in the clinic. Despite advances in molecular biology and immunotherapy, no significant improvement in prognosis for pancreatic cancer patients has been seen over the past 30 years. Tumours in the pancreas begin as pancreatic intraepithelial neoplasias (PanIN), and subsequent mutations cause them to progress to tumours ,2,3. TheRadiotherapy, chemotherapy and immunotherapies have all failed to improve survival, resulting in the worst 5-year prognosis of all common malignancies . 5-fluorregulatory cells, myeloid-derived suppressor cells (MDSC) which build crosstalk with TAM and suppress the effector T cells [One reason for the failure of treatments to provide therapeutic benefit in these patients has been postulated to be the low immunogenicity of pancreatic tumours due to their lack of neoantigens. This is because pancreatic carcinogenesis is driven by a low number of mutations typically ~3 mutations/Megabase ,18. Anot T cells ,33 and c T cells . The lat T cells , and can T cells ,41,42,43Clinical efforts to tackle pancreatic cancer by depleting the pancreatic tumour microenvironment have failed so far ,45,46, lThe cancer immunotherapy revolution has led to treatments that can provide clinical benefits in previously untreatable diseases. Several immunotherapy approaches, including immune checkpoint inhibitors (ICIs) ,50,51,52sata) or spatial peak temporal average (Ispta). There is an interplay between these parameters that determines whether thermal or mechanical effects predominate. At low frequencies and high-pressure amplitudes, mechanical effects  can be readily induced, whereas for higher frequencies and longer pulses thermal effects predominate. When focused ultrasound is applied in its continuous “thermal” mode (exposures lasting seconds), the tissue is subjected to frictional forces which are converted to heat [Therapeutic focused ultrasound is a physical modality that can induce localised thermal and mechanical effects non-invasively due to the compressions/decompressions induced when tissue is subjected to acoustic pressure waves . The imp to heat . Hyperth to heat ,85,86,87 to heat ,79,88,892 and, in the case of pulsing regimes, are in the order of 200 μs/800 μs “On time/Off time” [Histotripsy is the term used to describe focused ultrasound exposures that consist of high amplitude peak negative pressures (P–) (>15 MPa) applied to the tissue for milliseconds or less with the intention of inducing mechanical damage without significant temperature rises ,91,92,93ff time” ,103,104 Therapeutic focused ultrasound-mediated therapy has found several applications in the clinic owing to its multiple mechanisms of action and pleiotropic downstream biological effects. So far, these treatments have received FDA approval for the treatment of essential tremor, uterine fibroids, prostate cancer, osteoid osteoma and the palliation of pain from bone metastasis. It is also being investigated for its use in more than 150 clinical indications or disorders . Recentlparameters:43T = 45–56 °C, TID = 60 to >> 240 CEM) [cytotoxic cells against the tumours  [parameters:43T > 60 °C, TID > 5000 CEM) [The development of ultrasound and magnetic resonance imaging (MRI)-guided clinical systems capable of thermally ablating tissue led to the extensive investigation of the immunogenic effects of “thermal” focused ultrasound in preclinical and clinical studies . In vitr0 CEM43) ,114. In 0 CEM43)  and prod= 220 s) . In B16 0 CEM43) . These dcytotoxic and CD4+ T cells after HIFU treatment compared to before treatment  [helper cells (~3 fold increase) and Tcytotoxic (~2 fold increase) [Some of the earliest studies to investigate the immunological effects of HIFU in the clinic were those of Feng Wu and colleagues, who in 2004, reported the use of the JC ultrasound-guided HIFU system  to treat patients with osteosarcoma, hepatocellular carcinoma and renal cell carcinomas. One session of HIFU treatment was used to treat their primary tumours. Results showed blood increases of immune cells including T cells, T kW/cm2) . Subsequ kW/cm2) , lymphocncrease)  in the t+ cells in the treated and distant tumours were increased by “thermal” parameters, whereas “mechanical” parameters caused a decrease in FoxP3+ cells and kept the MDSC levels unaltered in distant tumours  [cytotoxic responses  [parameters:P– ~6 MPa, d.c. = 10%, prf = 5 Hz, pulses = 100) [regulatory cells, M1 and M2 macrophages were seen in 4T1 breast cancer cells. These effects could be associated with the induction of DNA damage by reactive oxygen species generated from the creation of cytosolic Ca2+ transients by the ultrasound treatments [h1, Tregulatory, Tcytotoxic, B cells, NK cells and macrophages in the TDLN of B16 and 4T1 tumours treated with the pHIFU parameters described above were seen [“Thermal” ablation of tissues can destroy cells and release cancer-associated antigens, but it is reasonable to hypothesize that at least some of these antigens may be heat-fixed from the extreme temperature rises (>56 °C) that are induced in tissues after HIFU treatment. This has led to suggestions that “mechanical” focused ultrasound may be better at releasing intact antigens than “thermal”. When the effects of “thermal” and “mechanical” focused ultrasound were combined with immunotherapy (CpG and anti-PD-1 antibodies), both induced innate immune responses. Here, FVB/n subjects carrying NDL tumour biopsies bilaterally were exposed to therapeutic focused ultrasound with the MR-guided Bruker BioSpec 7T small animal MR system. A 16-element 3 MHz annular array transducer (Imasonic SAS) was used for the sonications. Results showed that anti-immune MDSCs and FoxP3repeats) . Early sc. = 2%) ,118. Morc. = 2%) ,120,121.s = 100) . In addicytotoxic in B16GP33 tumour-bearing C57BL6 animals, as indicated by increases in their intracellular IFNγ expression. The histotripsy exposure of the B16-GP33 cells was so effective at stimulating cancer-specific lymphocyte responses that their stimulation approached that of direct exposure to the GP33 peptide  [parameters:P– = 14 MPa, prf = 1 Hz, d.c. = 1%, pulse duration = 10 ms long) [parameters:f = 1.5 MHz, P– =14 MPa, prf = 1 Hz, pulse duration = 13.3 ms, 15 s/focus) [cytotoxic after ultrasound-guided boiling histotripsy with the VIFU-2000 platform using a 1.5 MHz single-element, spherically-focused transducer, were also seen  [To test this hypothesis, treatment of melanoma tumours engineered to express a neo-antigen-mimicking peptide GP33 with histotripsy resulted in the activation of Tes = 50) . In addims long) . In-vivos/focus) . In renar focus) . These rr focus) .parameters:P– = 0.3 MPa, pulse duration = 10 ms, d.c. = 1%) [cytotoxic and the ratio of Tcytotoxic/Tregulatory, 3 and 18 days after treatment when compared to controls  [+ T cells, and by reducing anergy-related gene expression. LOFU increased HSP70, MHCII and B7 expression, and changed the tumoural distribution of calreticulin, suggesting a pro-immunogenic effect for these treatments. Moreover, there was increased maturation/activation of DC, further supporting the hypothesis that LOFU promoted T cell activation over anergy  [Low intensity focused ultrasound (LOFU) can also produce immune effects. Their biological basis is still uncertain, and the ultrasound exposure parameters need to be fine-tuned to produce the desired effect. This is particularly important in applications in the brain where a blood–brain barrier opening in the absence of a significant immune response is required. In this context, the NF-κβ pathway-associated inflammatory response and the infiltration of macrophages in the murine brain were seen after treatment with LOFU (c. = 1%) . In a stc. = 1%)  after exc. = 1%) . Treatmerepeats) . Further= 900 J) .Collectively, this information provides “proof of concept” that both “thermal” and “mechanical” focused ultrasound exposures can induce an immune response. The multiple physical mechanisms by which therapeutic focused ultrasound causes tissue disruption and disintegration can lead to different biological effects that may be tumour type- and tumour stage-dependent. Ultimately, our detailed understanding of these events should lead to the design of treatments aimed to create robust and durable anti-cancer immunological responses with higher anti-cancer efficiency and reduced side effects.More than 3500 pancreatic cancer patients have been treated with palliative intent with thermally ablative HIFU in the past 20 years. Reported studies showed that not only is the treatment of pancreatic tumours with HIFU feasible and safe but also that it produces effective pain relief, and in some cases, tumour growth control, improved survival rates and favourable clinical response. In addition, the feasibility of using “mechanical” focused ultrasound and LOFU to treat pancreatic cancer is under investigation preclinically.parameters:Power = 0.5–1.6 kW) [parameters:2Power = 10–15 kW/cm) [The earliest reports of HIFU-treated patients have come from China. In 2002 pain relief was reported in all 15 HIFU-treated patients  with late-stage pancreatic carcinoma. Their appetite, sleeping and mental health improved, and 5/15 patients gained weight. The tumours shrank in three cases and showed no change in the remaining 12. No serious complications  were seen, and amylase levels were unaltered in 13/15 patients. Mild pancreatitis was observed in two cases (–1.6 kW) . Another kW/cm2) . A retro kW/cm2) .Overall survival figures for pancreatic cancer patients have shown improvement after HIFU treatment. In 2011, the treatment of 13 stage III and 27 stage IV pancreatic cancer patients with ultrasound-guided HIFU  resulted in median overall survival of 10 months for patients with stage III disease, and 6 months for patients with stage IV disease. The one-year survival rate as a whole was 30% . In the parameters:Energy = 793,168 ± 704,778 kJ) [parameters:Power = 300 W for tumours < 4 cm in diameter, else Power = 400 W, Energy = 17 ± 12 kJ/mL) [parameters:Power = 100–400 W) [In Europe, the first report of patients treated with ultrasound-guided HIFU with palliative intent came from Italy. Six patients with pancreatic tumours of diameter 4.6 ± 1.4 cm were treated with HIFU ablation (JC HIFU system) using a 0.8 MHz, 20-cm diameter transducer, with a focal length of 15 cm. For these treatments, the patients followed a strict diet for 3 days and degassed water balloons were used to apply abdominal pressure to push the bowel out of the acoustic path. In all but 1 patient, the procedure was carried out with no complications and the 1-year survival rate and median survival after HIFU treatment were approximately 43% and 7 months, respectively  . In a se,778 kJ) . In Germ2 kJ/mL) . Finally0–400 W) . CollectHIFU has been combined with traditional oncologic interventions, such as chemotherapy and radiotherapy, to treat pancreatic cancer patients. Pain relief and minimal side effects have been observed in the majority of patients ,150,151.parameters:Treatment time = 114.5 ± 29 min, Sonication time = 19 ± 7 min, Power = 344 ± 72 W) [parameters:Treatment time = 110.5± 30.7 min, Sonication time = 18.4 ± 7.6 min, Energy = 386 ± 256 kJ) [In 2015 a report of a Spanish study of treatment of 43 patients with stage III or IV locally advanced pancreatic cancer with ultrasound-guided HIFU (JC HIFU system) and gemcitabine-based palliative chemotherapy demonstrated complete responses in 25% of patients, the overall median survival of 16 months, and a 33.5% survival of ~4 years. No deaths, emergency surgical procedures, large blood vessel rupture, nor gastrointestinal perforation were seen . In 2016 ± 72 W) . In a se 256 kJ) .parameters:Energy = 12 kJ/mL) [Case study reports have also demonstrated successful HIFU treatments. A 47-year-old patient with locally advanced unresectable pancreatic adenocarcinoma tumour in the body of the pancreas (4.0 cm × 3.2 cm) but with no metastasis in other organs was treated with 27 HIFU ablation sessions  to the tumour and nodal disease, in addition to six cycles of gemcitabine and capecitabine. The patient recovered fully after these treatments . Ultraso2 kJ/mL) . Recentl2 kJ/mL) .parameters:Treatment time = 124 ± 45 min, Sonication time = 936 ± 366 s, Energy = 343 ± 151 kJ) [No major complications resulting from HIFU treatments in pancreatic cancer patients have been reported. In 2011, third-degree burns and fistula formation between the tumour and duodenum were reported in a subset of 35 patients treated with ultrasound-guided HIFU (JC system) . A prosp 151 kJ) .parameters:sa = 1134 W/cm2, pulse duration = 500 msPower = 47.92 W, I) [In contrast, a retrospective investigation of the endocrine and exocrine function of the pancreas in 59 HIFU-treated patients with advanced pancreatic cancer found no change in amylase or glucose levels before or after treatment . In anot 500 ms) .To improve the safety and efficacy of HIFU treatments, methods of compensating for respiratory motion and improving thermometry have been developed, e.g., by fluid-filling the digestive tract surrounding the pancreas of patients ,169,170.parameters:Energy = 0.5–1 kJ, d.c. = 50%, prf = 3.3 Hz, pulses = 50–70) [So far, “mechanical” focused ultrasound treatments have received less clinical attention than “thermal”. The efficacy of the combination of concurrent gemcitabine and ultrasound-guided pHIFU (pHIFU FEP-BY™ HIFU unit) for the treatment of three patients with unresectable pancreatic cancer was compared to that of pHIFU treatments alone (nine patients). For the three patients who were successfully treated with gemcitabine and pHIFU, their overall survival ranged from 11 to 26 months. These three patients received a total of 32 treatments including six with pHIFU. Side effects included skin burns and mild abdominal pain, which were ultimately resolved. For the pHIFU-only treated patients, one case of pancreatitis was reported, and their median overall survival was ~10 months. Two cases of heat-induced subcutaneous sclerosis were also seen when more than 1000 J/lesion was delivered. In the 1st case, the injury was resolved within 4 weeks without treatment, but in the 2nd the injury remain unresolved 4 months later (= 50–70) .“low power” parameters:sata = 5.9 W/cm2, Ispta = 21.8 W/cm2, Energy = 15 W s, “high power” parameters: P– = 3.2 MPa, d.c. = 50%, prf = 40 Hz, duration = 10 s, Isata = 58.6 W/cm2, Ispta = 218.87 W/cm2, Energy = 50 W sP– = 3.2 MPa, d.c = 5%, prf = 1 Hz, duration = 30 s, I) [parameters:f = 1.2 MHz, Power = 7 W, d.c = 100%, T = 41.2 ± 1.3 °C) [Several preclinical studies have shown an improvement in drug delivery and anti-cancer effects in pancreatic tumours using pHIFU, sonoporation or sonodynamic therapy. In 2014, human Panc-1 tumours grown in nude Balb/c subjects were treated with “low” and “high” power focused ultrasound using an US-guided 1.1 MHz transducer . The combination of “low power” therapeutic focused ultrasound and gemcitabine induced higher levels (approx. ~1.5 fold) of necrosis, and apoptosis than unsonicated gemcitabine only treated, or “high power” therapeutic focused ultrasound treated subjects ( 50 W s) . In addi 50 W s) . In the  1.3 °C) . Additio 1.3 °C) , while t 1.3 °C) ,177, 5FU 1.3 °C) ,179, ant 1.3 °C) , gemcita 1.3 °C) , nanocom 1.3 °C) ,183,184, 1.3 °C)  and epir 1.3 °C) , have prThe benefits of immunotherapy have failed to materialise in patients with locally advanced or metastatic pancreatic cancer as has been shown in clinical trials involving anti-CTLA, anti-PD-1 and anti-PDL1 antibodies alone or in combination with gemcitabine, gemcitabine and nab-paclitaxel, anti-CCR4, tyrosine kinase inhibitors, anti-CXCR4, oncolytic viruses and vaccines (GVAX) ,199,200.+ cells and CD4+ cells compared to before HIFU treatment  [parameters:Treatment time = 124 ± 46 min, Sonication time = 932 ± 374 s, Energy = 343.9 ± 156.2 kJ, Power = 366 ± 91 W, Energy/Volume = 12.8 ± 4.5 kJ/mL) [parameters:Power = 103 W, Sonication duration = 752 s, 1st hyperechoic signal detected = 150 s.) [The first clinical study to report the immunological effects of HIFU in the pancreas was published in 2002. Increased natural killer cell activity and increases in the abundance of TIL were seen after “thermal” HIFU treatment. In this study, 15 patients with an average age of 62 years and tumour size on average of 5.6 cm participated and were treated with ablative “thermal” HIFU (FEP-BY01 system). The immune effects of these treatments were studied in the peripheral blood of 10 patients. Statistically significant increases of 25% in NK cells were seen, as well as non-statistically significant differences in CD31400 kW) . Recentl5 kJ/mL) . In a ca 150 s.) . In anot 150 s.) .+ cells) and regulatory T cells  and an increase in DC (as a percentage of CD45+ cells) was seen 14 days after histotripsy treatments. The same authors also showed the release of DAMPS, including HMGB1 release, after the treatment of the Pan02 cells in vitro with the same histotripsy parameters  [2 nanoparticles were administrated systemically in animals bearing syngeneic bilateral KPC tumours, sonodynamic therapy with a SP100 Sonidel sonoporator  increased Tcytotoxic and decreased Tregulatory in both target and off-target tumours and induced the abscopal effect in off-target tumours  [+ T cells and Tcytotoxic cell infiltration in the off-target tumour of the treated animals was increased by the sonodynamic therapy  [cytotoxic, IFNγ+ Tcytotoxic cells, and the ratio of these cells to Tregulatory and MDSC in the tumours of the animals treated with pHIFU and ICI compared to the control animals. These results suggested that treatment of pancreatic tumours with pHIFU can enhance ICI anti-cancer therapy by changing the immune contexture of tumours  [In preclinical studies, histotripsy treatment of approximately 60–75% of subcutaneous murine Pan02 tumours with an ultrasound-guided transducer induced a cavitation cloud, confirmed by ultrasound imaging, tumour destruction and a decrease in tumour size. No changes in immune cells in the microenvironment of the tumours were seen 24 h and 7 days after histotripsy, and then a decrease in macrophages (percentage of CD45 250 Hz) . Sonodyn 100 Hz) . In anotjection) . Focused= 10 ms) .Despite encouraging clinical results and the lack of severe adverse effects from therapeutic focused ultrasound treatments, no randomised controlled trial to test whether focused ultrasound can provide a significant survival benefit to patients with pancreatic cancer has ever been undertaken. The FDA approval of ICI for a small minority of pancreatic cancer patients (~1%) with high microsatellite-instability in their genomes ,207 provStiff pancreatic tumours are excellent candidates for treatment with physical modalities, such as therapeutic focused ultrasound. Strong evidence is now in place, both preclinically and clinically, that therapeutic focused ultrasound has a significant benefit in the treatment of pancreatic cancer. Progress is hindered, in part, by the high cost of some immunotherapies, although ICI antibody patent release in the following years would be expected to lower the associated costs. Furthermore, technological developments  are expected to lead to whole tumour ablation in most cases. The development of mechanical HIFU for clinical use will offer greater pro-inflammatory effects in patients, as has been shown preclinically. The establishment of an ultrasound thermal and cavitational dose will improve our understanding of the bioeffects of focused ultrasound and provide the capability for clinicians to standardise treatment between different centres. Ultimately the rapid growth in the use of therapeutic focused ultrasound in the clinic will continue, and in the following years, its increased use in combinatorial treatment protocols for pancreatic cancer patients will be seen.


RB1 gene germline mutations and its influence on clinical decisions is minimally explored.India accounts for 20% of the global retinoblastoma (RB) burden. However, the existing data on RB1 gene mutations was performed through next-generation sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. The mutation and non-mutation groups were compared for clinical parameters especially severity, progression and recurrence.Fifty children with RB underwent complete clinical examination and appropriate multidisciplinary management. Screening of germline RB1 variations in 29 probands, inclusive of 3 novel mutations, known 16 mutations and heterozygous whole gene deletions. The mutation detection rate (MDR) was 86.2% in BLRB and 19% in ULRB. Associations of disease recurrence (p = 0.021), progression (p = 0.000) and higher percentage of optic nerve invasion, subretinal seeds and high-risk pathological factors were observed in the mutation group. Clinical management was influenced by the presence of germline mutations, particularly while deciding on enucleation, frequency of periodic follow up and radiotherapy.Twenty-nine patients had bilateral RB (BLRB) and 21 had unilateral RB (ULRB). The genetic analysis revealed 20 RB1 mutations, and our mutation detection rate was on par with the previous global studies. In our study, genetic results influenced clinical management and we suggest that it should be an essential and integral component of RB-care in India and elsewhere.We identified novel  RB1gene, owing to homozygous allelic mutations, loss of heterozygosity mechanism or gene silencing [RB1 is a nuclear phosphoprotein, essential for G1/S check point during the cell cycle regulation, while in a dephosphorylated state binds to mitotic agents like E2F, viral particles and other factors, but releases them during mitosis when phosphorylated. RB1 gene is located on chromosome band 13q14.2, consisting of 27 exons, which encodes a 4.7 kb mRNA. So far, 1748 unique RB1 variants in 3366 individuals have been identified and summarized in the Leiden Open Variation Database (LOVD) [RB1 mutations are unique and found in exon, splicing introns and untranslated regions [RB1 exon deletions are seen not only in RB but also less frequently in breast cancer, osteosarcoma and lung cancer.Retinoblastoma (RB) OMIM#180200) is the commonest childhood intraocular tumor, with a global estimated annual incidence of 1 in 15,000–20,000 live births 80200 is . RB occuRB1 mutations, who may need genetic screening [Usually, in any given population, there are more children with unilateral RB (ULRB-60%) than bilateral (BLRB-40%) and a clinician has to be noted that a majority of those with BLRB and a small proportion of those with ULRB might have germline creening . Geneticcreening .RB1 mutations have been reported from various populations around the world like, Argentina [Unless genetic testing is available, the minority of unilateral hereditary cases, fail to get the desirable clinical management decisions and frequent clinical surveillance. Hereditary RB tends to be early in onset, bilateral and multifocal, hence needs continuous surveillance for effective management. All cases with mutation, as mentioned earlier, have a lifetime risk for osteosarcoma, soft tissue sarcoma, malignant melanoma or multiple brain tumours. Hence they need lifelong follow-ups, as opposed to sporadic cases, which may not have genetic predisposition. Between 1905 and 2005 about 199 RB survivors were retrospectively analysed for second primary tumours (SPT) and found that 44 of them developed SPT [rgentina , Brazil rgentina , China [rgentina , 13, Colrgentina , Ecuadorrgentina , Egypt [rgentina , India [rgentina –21, Iranrgentina , Israel rgentina , 24, Itargentina , Korea [rgentina , Netherlrgentina , Spain [rgentina , 29, Malrgentina , Mexico rgentina , Moroccorgentina , New Zeargentina , Pakistargentina , Swiss [rgentina , Tunisiargentina  Singaporrgentina  Thailandrgentina  and Unitrgentina . Out of rgentina .RB1 gene mutations across the globe, including India, there is limited information on how the genetic result could influence clinical management outcomes. Hence, we undertook this study to describe and correlate the genetic and clinical parameters of 50 RB patients from India. We also examined the opportunities and challenges in clinical decisions which were influenced through RB1 gene screening in a developing country scenario.Though enormous number of studies are available on RB1 mutations.Fifty (48 unrelated and two related siblings) RB patients (aged 0.2–5.3 years) with various clinical presentations, from the Department of Paediatric Ophthalmology, Narayana Nethralaya, Bangalore, India were recruited from June 2014 to Feb 2015. Among these twenty-nine were BLRB and twenty-one had ULRB. A complete clinical examination was carried out under general anaesthesia which included dilated retinal evaluation, imaging of retina using wide field fundus camera (Retcam), measurement of intraocular pressure, anterior segment evaluation by handheld slit lamp. Also, magnetic resonance imaging (MRI) of the orbits and brain, B scan ultrasonography of the eye, cerebrospinal fluid analysis and bone marrow analysis were performed when indicated. The clinical disease was classified as per the AJCC TNM classification for RB, as well as the International Classification of Intraocular Retinoblastoma . The stuNucleospin Blood XL kit (Macherey–Nagel)—About 5 of peripheral blood from the child is collected and 500 μl of proteinase K and PBS are added for lysis of RBCs. Then 10 ml of buffer BQ1 is added a shaken vigorously for 2 min and incubated at 56 °C for 15 min. Then add 10 ml of 96–100% ethanol and vortex for 10 s for lysate formation. Take Nucleospin Blood XL column and add 15 ml of the lysate and centrifuge at 5000 rpm for 3 min. Discard the flow-through and repeat the last step. Add 7.5 ml Buffer BQ2 and centrifuge at 5000 rpm for 2 min and repeat the step for 20 min. Insert the column into new collection tube and add 750 μl of prewarmed 70 °C Elution Buffer BE and incubate at room temperature for 16 h. The last step may be repeated and when centrifuged at 5000 rpm for 5 min, highly pure genomic DNA elutes through the silica membrane.RB1gene for 72 h and extracted using streptavidin beads, washed and normalized. The libraries were then sequenced to mean > 80–100× coverage on Illumina sequencing platform (HiSeq 2500). The sequences obtained are aligned to human reference genome (GRCh37/hg19) using BWA program [Genomic DNA was used for targeted gene capture using a custom capture kit. Briefly, 1ug of DNA was subjected to fragmentation resulting in an average size of 150 bp followed by end repair, adenylation, adaptor ligation and amplification to obtain whole genome libraries using the Kapa DNA library preparation kit v2.14. These libraries were then hybridized to biotinylated probes  specific to  program , 43 and  program , 45 to i program . Clinica program –54.RB1 gene, we performed Multiplex Ligation-dependent Probe Amplification (MLPA). SALSA MLPA kit P047 RB1  was used as per manufacturer’s recommendations.In order to detect large deletions/duplications in the Multivariate analysis for genotype phenotype correlation was done using Pearson Chi square test, SPSS software. Clinical factors like sub retinal seeds, optic nerve invasion, pathological high-risk factors (HRF), tumour recurrence, tumor resistance to treatment, need for 2nd line drugs like topotecan and need for radiotherapy were analysed in the mutation versus no mutation groups.RB1 gene variations in 29 probands, inclusive of three novel mutations  (intronic splice variant) (ClinVar ID: SCV001571344.1), Q444P (ClinVar ID: SCV001571345.1) and S567P (ClinVar ID: SCV001571346.1)), previously reported 16 mutations (22 probands: 44%) and heterozygous deletion of whole RB1 gene . The types of mutations were, non-sense being the maximum [Of 50 RB patients, 29 had BLRB (average age at presentation of 1.8 years) and 21 had ULRB (average age at presentation of 2.3 years). A family history of RB was observed in two patients. In the BLRB group, 25 out of 29 probands (86.2%) had a germline mutation whereas in the ULRB group, 4 out of 21 (19%) had a mutation. NGS and MLPA analyses revealed total of 20 p = 0.021 and p = 0.000 respectively). Notably, of the total 10 recurrences in the current cohort, 9 patients had the mutation  was identified in two unrelated patients with bilateral RB. The novel nucleotide changes include two missense substitutions—c.1699 T > C (p.S567P), c.1331A > C (p.Q444P) and one splice site variation (c.1050-8_1050-2delTTATTTA). Bioinformatics prediction analysis of SIFT, PolyPhen–2, Provean, showed that the missense substitutions  had deleterious effect which may affect the functional properties of the protein and both the missense variations are present in the retinoblastoma-associated protein A domain of the RB1 protein. The three novel mutations were in BLRB patients. One of the BLRB patients, who presented at 2.5 years of age, had a p.W78X mutation and he was diagnosed to have pinealoma and was a case of trilateral RB. Another proband, presented with bilateral disease at 1.5 years with a positive family history. The father had regressed tumour, both the daughter and father carried the same familial mutation, c.1789C > T (Q597Ter). Interestingly, the half sibling of this proband, who was of the same father, presented at the age of 2.3 years with BLRB and had the same mutation c.1789 C > T (Q597Ter). Another interesting aspect was the varied clinical spectrum presentation and outcome of our four BLRB probands who all had the same termination mutation c.1333C > T (p.R445Ter) . In our study, we found 20 RB1 gene variations in 29 probands (79%), inclusive of three novel mutations, 16 previously reported mutations, four heterozygous deletions of the whole RB1 gene. We had one case each of frameshift and commonly reported R251X, R320X mutations and it is to be noted that those with the arginine/termination mutations have a risk to develop SPT [MYCN gene mutations, which we did not study. Mosaicism is a tricky issue in RB diagnostics and prenatal genetic counselling, hence may go unnoticed suggests Rushlow et al. [The mutation detection rates across countries in BLRB varied from 100 to 16.6% and in ULRB from 56.3 to 9.5% , lack of hotspot and the variable intronic lengths . Three pThere are many limitations of our study, we did not analyse tumor DNA samples and hence we did not detect somatic mutations especially in non-hereditary retinoblastoma . Also, tRB1 mutations using targeted NGS and MLPA methodologies, which found detection rates on par with most global studies. Comparing case-wise genetic findings with various clinical parameters and mutations found that there were clinical phenotypic and allelic heterogeneities. The mutation group had a higher clinical risk of recurrence, which influenced clinical management. RB1 mutation screening is an important tool in RB-care globally, including developing countries.In summary, 50 RB patients were screened for


Monodelphis domestica. Opossums are unique in their ability to fully regenerate their CNS after an injury during their early postnatal development. Thus, we used cortex of postnatal day (P) 3–5 opossum to establish long-surviving and nearly pure neuronal cultures, as well as mixed cultures composed of radial glia cells (RGCs) in which their neurogenic and gliogenic potential was confirmed. Both types of cultures can survive for more than 1 month in vitro. We also prepared neuronal cultures from the P16–18 opossum cortex, which were composed of astrocytes and microglia, in addition to neurons. The long-surviving opossum primary dissociated neuronal cultures represent a novel mammalian in vitro platform particularly useful to study CNS development and regeneration.Primary dissociated neuronal cultures have become a standard model for studying central nervous system (CNS) development. Such cultures are predominantly prepared from the hippocampus or cortex of rodents (mice and rats), while other mammals are less used. Here, we describe the establishment and extensive characterization of the primary dissociated neuronal cultures derived from the cortex of the gray South American short-tailed opossums,  A full understanding of the structure, function, and development of the mammalian central nervous system (CNS) is necessary to develop treatments that could allow successful regeneration of the adult injured or degenerated tissue. Mammalian developing CNS is challenging to study because of the extreme complexity of the dynamic events involved, including cell proliferation, migration, and differentiation. Moreover, it emerged that neurodegenerative diseases often originate during development, despite late onset . Opossums are marsupials (Metatheria), which are mammalian infraclass that diverged from placental mammals (Eutheria) between 170 and 190 M years ago  are born after 14 days of gestation at a very immature state  of different ages, in which different CNS cell types predominate, including neurons, radial glia cells (RGCs), and astrocytes. We have chosen P3–5 and P16–18 opossums since those ages differ essentially in their capacity to regenerate spinal cord tissue after injury. Our protocols allow long-term survival of neuronal cell cultures (up to 1 month), as well as efficient maintenance and differentiation of RGCs into neurons in vitro. These M. domestica-derived primary CNS cultures are novel and robust in vitro platforms to use in a variety of studies, including development and regeneration.Here, we describe the protocols to successfully develop and establish different Monodelphis domestica) pups of both sexes at postnatal day (P)3–5 and 16–18 were used. The M. domestica colony was maintained at the animal house facility of the University of Trieste, following the guidelines of the Italian Animal Welfare Act, and their use was approved by the Local Veterinary Service, the Ethics Committee board, and the National Ministry of Health (Permit Number: 1FF80.N.9Q3), following the European Union guidelines for animal care . The animals are housed in standard laboratory cages in a temperature- and humidity-controlled environment  with a 12/12 h light/dark cycle and ad libitum access to food and water. According to recently reported developmental transcriptome analysis  dissection solution, consisted of 113 mM NaCl, 4.5 mM KCl, 1 mM MgCl2 × 6H2O, 25 mM NaHCO3, 1 mM NaH2PO4, 2 mM CaCl2 × 2H2O, 11 mM glucose and 0.5% w/v Penicillin/Streptomycin/Amphotericin B, pH 7.4 . The meninges were removed, and the tissue was chopped into small pieces. Before enzymatic digestion, the cortices were washed three times with the sterile phosphate-buffered saline (PBS) containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 . Cortices were digested with prewarmed trypsin in PBS  by incubating at 32.5°C. The tissue from P3 to P5 pups was incubated in 0.5% w/v trypsin for 10 min, while that of P16–18 pups was incubated in 2.5% w/v trypsin for 15 min. After three washes with PBS, cells were dissociated using a 1 ml tip by pipetting up and down 15–20 times in a trituration solution containing 10 μg/ml DNAse I (Sigma–Aldrich), 1 mg/ml trypsin inhibitor (SCBT), and 1% w/v bovine serum albumin  in Hank’s Balanced Salt Solution (HBSS) solution, w/o Ca2+ and Mg2+ (Pan-Biotech). The supernatant containing dissociated cells was collected and layered on top of the 5% w/v BSA cushion in HBSS in the 5 ml tube to remove the cell debris. The trituration step was repeated two times for P3–5 and three times for P16–18 digested cortices. Cells were collected by centrifugation for 5 min at 100 g and then resuspended in a plating medium consisting of Dulbecco’s minimum essential medium (DMEM) with stable glutamine supplemented with 10% w/v fetal bovine serum (FBS) and 1% w/v Penicillin/Streptomycin  and purified by preplating the cell suspension for 5 min on the plastic tissue culture dishes. Cells were counted using the hemocytometer and plated on glass coverslips (12 mm diameter) precoated with 50 μg/ml poly-L-ornithine and 2 μg/ml laminin  at the density of 1 × 105 cells per well in a 12-well plate or 5 × 104 cells per well in a 24-well plate. The next day, 2/3 of the medium was changed with the neuronal medium containing Neurobasal medium supplemented with B27 , 1 mM L-glutamine, and 1% Penicillin/Streptomycin (both from Pan-Biotech). Subsequent media changes were done once per week changing only a half of the medium with the fresh neuronal medium. The cortical cultures were maintained in an incubator at 32°C, 5% CO2, and 95% relative humidity.The dissociation protocol was developed following the procedures previously described for rodents  and then transferred to poly-L-ornithine and laminin-coated glass coverslips in neuronal medium  to differentiate over the next 7 days.Radial glial cells (RGCs) were prepared using the same protocol for dissection and dissociation as described for P3–5 neuronal cultures. Unlike neuronal cultures, RGCs were plated on poly-L-ornithine-coated dishes or coverslips without laminin at the density of 5 × 10® 555 , goat anti-mouse Alexa Fluor® 488 , goat anti-rabbit Alexa Fluor® 555 , goat anti-rabbit Alexa Fluor® 647 , goat anti-mouse immunoglobulin (Ig) G1 Alexa Fluor® 488  and goat anti-mouse IgG2a Alexa Fluor® 555  and the incubation time was 30 min in the dark. For F-actin staining, Phalloidin-iFluor 488  was incubated for 30 min together with the secondary antibody. Cell nuclei were stained with a 300 nM nuclear stain 4′,6-diamidino-2-phenylindole  diluted in PBS for 5 min. Finally, coverslips were washed with PBS and in dH2O. Coverslips were mounted on a glass slide using the mounting medium  and sealed with nail polish. All the incubations were performed at RT. Proliferation assay was done using Click-iTTM 5-ethynyl-2′-deoxyuridine (EdU) Cell Proliferation Kit, Alexa FluorTM 488 dye (Thermo Fisher Scientific). EdU was added to the cells according to manufacturer instruction immediately after the plating and incubated for 24 h. Cells were fixed at DIV1 and immunolabeled for 30 min at RT in Click-iT® reaction cocktail. The cells were analyzed using an Olympus IX83 inverted fluorescent microscope  equipped with differential interference contrast (DIC) and fluorescence optics  and Cy5 . Fluorescence images were acquired with Hamamatsu Orca R2 CCD camera  and CellSens software . 10× 0.3 numerical aperture (NA) air, 20× 0.5 NA air, and 40× 1.4 NA oil immersion objectives were used. For each image 15–30 slices were acquired with slice spacing of 1 μm (10× and 20× objectives) and 0.3–0.5 μm (40× objective). For each image, a maximum intensity projection was used. CellSens and ImageJ by W. Rasband  with 4% paraformaldehyde  containing 200 mM sucrose in PBS, pH 6.9. After fixation, cells were washed with PBS, saturated with 0.1 M glycine, permeabilized with 0.1% Triton X-100  in PBS, and lastly washed with PBS, each step lasting 5 min. Cells were blocked with 0.5% w/v BSA (Pan-Biotech) in PBS for 30 min. Incubation with the primary antibodies  was doneT-test was used when comparing two normally distributed data groups with equal variances, and in case of unequal variances, Welch’s t-test was used. The accepted level of significance was p < 0.05. p < 0.001 very significant ***, 0.001–0.01 very significant **, 0.01–0.05 significant *, ≥0.05 not significant.All results have been obtained from at least three independent experiments and are presented as a bar graph with mean ± SEM. Statistical analysis was performed using GraphPad Prism 8.4 . To test the normality of data, depending on the number of values tested, either D’Agostino–Pearson or Shapiro–Wilk normality test was used. Brown–Forsythe test was used to test the equality of variances. One-way ANOVA was used to compare three or more data groups when data followed Gaussian distribution. Following the one-way ANOVA, the Holm–Šídák test was performed for multiple comparisons between data groups. Data groups with different variances that fail a normality test were compared using the Kruskal–Wallis test. Following the Kruskal–Wallis test, Dunn’s multiple comparisons test was performed for multiple comparisons between data groups. As described in “Materials and Methods” section in detail, the dissection protocol was adapted from the standard and well-established protocols for rodent embryonic or postnatal neuronal cultures from cortex or hippocampus  and were positive for β-tubulin III marker  fluorescence stain (2 (n = 40). To the best of our knowledge, these are the first experimental observations showing that neuronal GCs derived from the opossum cortex can be efficiently obtained in vitro, with a size comparable to rat hippocampal GCs  were triple positive for TUJ1, MAP2, and NeuN  was expressing only NeuN . Finally, triple-negative non-neuronal cells (TUJ1−/MAP2−/NeuN−) were 2.28 ± 0.35% of all cells . Neurons positive only to NeuN marker (TUJ1−/MAP2−/NeuN+ cells) were reduced to 4.53 ± 0.55% (n = 442). However, at DIV4 the total number of neurons  exceeded hugely the number of non-neuronal cells, which was further reduced to around 2% , indicating the progression of neuronal differentiation in culture  neurons present in culture coexpressed TUJ1 and MAP2, while only a minor portion was made up by TUJ1-negative neurons, expressing MAP2 and NeuN markers  while at DIV4 it reached 503.10 ± 11.15 μm2 (n = 95). Nuclei surface increased 1.8-fold: from 140.93 ± 3.01 μm2 (n = 83) at DIV1–261.03 ± 4.77 μm2 (n = 95) at DIV4. This analysis was done using MAP2 and DAPI staining for soma and nuclei, respectively . Our in vitro observations correlate with the increase in average neuron size observed in vivo, during the first postnatal week in the rat brain  and for all time points considered (n = 603) was reached (n = 980) of cells were TUJ1+ neurons  of GFAP+ cells in cultures derived from P16 to P18 cortex. In addition to GFAP, we confirmed the expression of intermediate filament protein vimentin , the most commonly used marker of astrocytes, which in n = 531, In addition to astrocytes, Iba1-positive microglial cells were found at DIV1 in cultures derived from the P18 cortex, accounting for 2.15 ± 0.71% of cells n = 531, . We were+ cells .To maintain proliferative cell state, instead of using serum-free Neurobasal/B27 medium . Five days after replating, TUJ1-positive neurons were found  and that with the use of the animals of different postnatal age, as well of different procedures and media, the enrichment of different CNS cell types, such as neurons or RGCc, can be obtained. Moreover, we have shown that both neuronal and astrocyte differentiation, the formation of GCs, synapses, and neurospheres as well as RGCs proliferation occurs in vitro, which makes opossum cortical cultures a favorable platform for investigating CNS development and regeneration.In this study, we have shown that long-term primary neuronal cultures can be successfully obtained from neonatal opossums  neuronal cultures have been obtained at DIV4, resembling E16–18 mice or rat cortical cultures with >90% neurons . We showed that this was due to the increased ratio of non-neuronal cells, by using specific markers for astrocytes and microglia. These results are comparable to postnatal (P0–3) rodent cultures, where the percentage of glia can vary roughly between 10% and 20%  cultures, the formation of 3D- and organoid-like structures  opens thM. domestica and other mammalian species, especially human proteins, should allow, facilitate and encourage neuroscientists for future investigations on opossums.The high degree of protein sequence homology (Mikkelsen et al., The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Ethical Committee of the Department of Biotechnology of the University of Rijeka.JB and MM designed and supervised the research. AP, IT, MP, MI, and SM prepared primary cultures. AP, JB, MP, MI, and SM performed immunofluorescence and imaging. AP, JB, MP, and MI analyzed the data. All authors contributed to the article and approved the submitted version.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.


The Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs), namely essential thrombocythaemia (ET), polycythaemia vera (PV) and primary myelofibrosis (PMF), are a group of chronic clonal haematopoietic disorders that have the propensity to advance into bone marrow failure or acute myeloid leukaemia; often resulting in fatality. Although driver mutations have been identified in these MPNs, subtype-specific markers of the disease have yet to be discovered. Next-generation sequencing (NGS) technology can potentially improve the clinical management of MPNs by allowing for the simultaneous screening of many disease-associated genes.The performance of a custom, in-house designed 22-gene NGS panel was technically validated using reference standards across two independent replicate runs. The panel was subsequently used to screen a total of 10 clinical MPN samples . The resulting NGS data was then analysed via a bioinformatics pipeline.The custom NGS panel had a detection limit of 1% variant allele frequency (VAF). A total of 20 unique variants with VAFs above 5%  and one pathogenic variant with a VAF of between 1 and 5% were identified across all of the clinical MPN samples. All single nucleotide variants with VAFs ≥ 15% were confirmed via Sanger sequencing.MPL.The high fidelity of the NGS analysis and the identification of known and novel variants in this study cohort support its potential clinical utility in the management of MPNs. However, further optimisation is needed to avoid false negatives in regions with low sequencing coverage, especially for the detection of driver mutations in The online version contains supplementary material available at 10.1186/s12920-021-01145-0. Myeloproliferative neoplasms (MPNs) are a group of chronic clonal haematopoietic disorders. The MPN subtypes include the Philadelphia (Ph) chromosome-positive chronic myeloid leukaemia, as well as the Ph-negative MPNs such as essential thrombocythaemia (ET) (characterised by an overproduction of platelets), polycythaemia vera (PV) (characterised by an overproduction of red blood cells), and primary myelofibrosis (PMF)  and a late, overt fibrotic stage (overt-PMF)). Among the three, ET is the most indolent, whereas PMF is associated with the highest symptom burden and worst prognosis. Common causes of morbidity and mortality include thromboembolic and/or haemorrhagic complications, as well as disease progression to myelofibrosis (MF) and/or transformation to acute myeloid leukaemia (AML), all of which vary in frequencies between MPN subtypes . To dateJAK2, CALR and MPL genes contributed towards the improved accuracy of MPN diagnostics. The JAK2 gene encodes the JAK2 non-receptor tyrosine kinase associated with several receptors which are critical for normal myelopoiesis, including the erythropoietin, thrombopoietin, and granulocyte colony-stimulating factor receptors; the MPL gene encodes the thrombopoietin receptor; while the CALR gene encodes the chaperone protein calreticulin. Driver mutations in these genes  lead to the constitutive activation of associated receptors and the subsequent amplification of downstream signalling pathways, which result in the aberrant cell proliferation in MPN. However, these driver mutations are not MPN subtype-specific, and can also be found in other myeloid diseases such as myelodysplastic syndromes (MDS), myelodysplastic/myeloproliferative neoplasms (MDS/MPN) and AML [The discovery of driver mutations in the  and AML , 5. In aThe management of MPNs requires an integrated, multimodal approach that includes the evaluation of clinical features, peripheral blood smear, bone marrow morphology, immunophenotype, cytogenetics, as well as molecular genetics, as recommended in the 2016 World Health Organisation (WHO) classification guidelines . As MPNsASXL1, EZH2, TET2, IDH1, IDH2, SRSF2 and SF3B1, contributing towards improved disease prognostication. In a novel prognostic model developed by Grinfeld, et al. [Rapid advancements in gene sequencing technology in the last decade have led to the discovery of other MPN-associated genetic aberrations, such as mutations in , et al. , patient, et al. . HoweverABL1, ASXL1, CALR, CBL, CEBPA, CSF3R, DNMT3A, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, MPL, NPM1, PDGFRA, RUNX1, SF3B1, SRSF2, TET2, TP53, and U2AF1) by using a set of reference standards and a small cohort of clinical MPN samples.In this study, we report on the validation of a custom NGS panel which targets 22 MPN-associated genes , Universiti Kebangsaan Malaysia Medical Centre Research Ethics Committee (UKM FPR.4/244/FF-2018-420), Medical Research & Ethics Committee Ministry of Health Malaysia (MREC 8KKM/NIHSEC/P-19-99), and Sunway University Research Ethics Committee (PGSUREC 2019/010).ABL1, ASXL1, CALR, CBL, CEBPA, CSF3R, DNMT3A, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, MPL, NPM1, PDGFRA, RUNX1, SF3B1, SRSF2, TET2, TP53, and U2AF1  to target the hotspot exons of 22 genes known to be frequently mutated in MPNs, namely FLT3 (p.ΔI836), IDH1 (p.R132C), and JAK2 (p.V617F); 3) Tru-Q7 (1.3% Tier) Reference Standard (Horizon)—as a positive control for 1.3% mutant allele in FLT3 , IDH1 (p.R132C/H), IDH2 , JAK2 (p.V617F), KIT (p.D816V), and PDGFRA (p.D842V); and 4) Seraseq™ Myeloid Mutation DNA Mix —as a positive control for mutant allele in ABL1 (p.T315I), ASXL1 , CALR (p.L367fs*46), CBL , CEBPA , CSF3R (p.T618I), FLT3 , IDH1 (p.R132C), JAK2 , MPL (p.W515L), NPM1 (p.W288fs*12), SF3B1 , SRSF2 (p.P95_R102del), and U2AF1 (p.S34F)  according to the manufacturer’s guidelines. The purity (A260/A280 and A260/A230) and concentration of extracted DNA were assessed using the Nanodrop™  spectrophotometer. DNA samples with satisfactory purity ratios of 1.80–2.00 (A260/A280) and 2.00–2.20 (A260/A230) were visualised using 1% agarose gel electrophoresis and stored for downstream experiments.Genomic DNA was extracted from each blood sample using the MaxwellThe NGS libraries were prepared according to the AmpliSeq for Illumina On-Demand, Custom and Community Panels Reference Guide Protocol, DNA Panels Standard Workflow Procedure for Three Primer Pools, using reagents from the AmpliSeq™ Library PLUS for Illumina®. The concentration of the input DNA was measured using the Qubit dsDNA BR Assay and the concentration of the constructed libraries was assessed using the Qubit dsDNA HS Assay on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) following the manufacturer’s protocol. The quality of the constructed libraries was assessed using the DNA Hi Sens Lab Chip  according to the DNA High Sensitivity Assay User Guide for LabChip GX Touch/GXII Touch Standard Sample Workflow. Libraries were denatured and diluted according to the MiSeq System Denature and Dilute Libraries Guide Protocol A: Standard Normalization Method for MiSeq Reagent Kit v2, with a PhiX library used as a sequencing control. Pooled sequencing of 6 samples per run was conducted on an Illumina Miseq using the MiSeq Reagent Micro Kit v2 (2 × 150 base pair (bp), paired end) to a minimum coverage depth of 5000 × according to the manufacturer’s protocol.For the technical validation of the custom NGS panel, only variants that were present in the four reference standards were filtered-in and prioritized; whereas for the screening of the clinical MPN samples, all annotated variants were manually reviewed by filtering and prioritizing using the following criteria: (1) all variants except for exonic variants were excluded, (2) variants with minor allele frequencies (MAFs) of ≥ 1%  were excluded, and (3) potential sequencing errors (variants with VAF of < 5% and/or appear in majority of the samples) were excluded. In addition, variants with VAFs between 1 and 5% were inspected to check for any pathogenic/likely pathogenic variants. Aligned read (.bam) files for all samples were then manually inspected to confirm the presence of the filtered-in and prioritized variants using the Integrative Genomics Viewer (IGV) software . In order to identify any putative novel variants, the variants were manually checked against dbSNP  as well TET2 exon 7 p.D1314Mfs*48 variant, the 360 GC Enhancer in the PCR mixture was found to reduce the PCR product yield, and was therefore replaced with nuclease free water. The PCR thermocycling conditions for all NGS detected variants were as follows: 95 °C for 10 min, 35 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, followed by a final extension at 72 °C for 7 min. The PCR products were visualised using a 1% agarose gel electrophoresis to confirm successful amplification and then purified using Wizard® SV Gel and PCR Clean-Up System (Promega) by centrifugation according to the manufacturer’s instructions but eluted in 15 mL of nuclease free water twice to obtain higher product concentration. The concentration and purity of the PCR products were assessed using the Nanodrop™ spectrophotometer as previously described. Samples with satisfactory concentration and purity ratios were sent for Sanger sequencing. Alignment of Sanger sequence (.seq) files were conducted via the ClustalW algorithm using the Jalview software [The Ensembl genome browser  and NCBIsoftware  whereas 2, > 93% of clusters passed the quality filter, and > 95% of the read bases with quality scores of above Q30, which were close to The Miseq System specifications of 865–965 K/mm2 cluster density and > 80% of bases above Q30  and AMPL1156 , while AMPL117202  had a coverage depth of below 100x  were recruited for this study , Ampang Hospital (n = 1) and Universiti Kebangsaan Malaysia Medical Centre (n = 2)) , ASXL1 (n = 4), DNMT3A (n = 1); RUNX1 (n = 1), SF3B1 (n = 1), TET2 (n = 6), and U2AF1 (n = 2)  was reported in dbSNP (rs144448357). However, it is unknown whether it was previously identified in MPN due to the lack of ClinVar and COSMIC data (Table JAK2 V617F mutation. The patient presented with increased platelet (859 × 109/L) and white cell counts (17.8 × 109/L), as well as constitutional symptoms  stopgain variant  was found to be located in the same dbSNP cluster rs750318549 as the previously reported ASXL1 p.Gly646Trpfs*12 (c.1934dupG) stopgain, which is the most common ASXL1 mutation accounting for > 50% of all identified ASXL1 mutations in myeloid malignancies [ASXL1 p.Gly646Trpfs*12 is the result of a duplication of a G nucleotide within a homopolymer region of eight G nucleotides [ASXL1 p.Gly646Trpfs*10 identified in this study is the result of an insertion of 67 nucleotides at position chr20: 31022442, making it a novel stopgain variant. Two other ASXL1 variants identified in this study, ASXL1 p.Leu731Tyrfs*12  and Q1433Q  were also putative novel variants with no dbSNP, COSMIC or ClinVar data; whereas the ASXL1 p.Tyr591*variant  has been reported in various diseases including ET and MF [Four T and MF , chronicT and MF , mast ceT and MF , as wellT and MF , but hasTET2 variants were identified in this study, of which, TET2 p.Tyr1245Cys  was not found to be reported in dbSNP, COSMIC or the ClinVar database. The TET2 p.Ala304Val  and p.Phe868Leu  variants have not been previously reported in MPN. TET2 p.Ala304Val has only been previously identified in melanoma [TET2 p.Phe868Leu has been previously identified in estrogen- and progesterone-receptor positive breast cancer [TET2 p.Asp1314Metfs*48 variant  has been reported in ET [TET2 p.Ile1195Val (rs568009712)  and p.Glu1513Gly (rs553669299) , but the associated disease(s) are unknown. Of note, Sample 02 was found to carry another TET2 variant—the p.Tyr1631* stopgain, reported as pathogenic in the COSMIC database and previously identified in angioimmunoblastic T cell lymphoma [Seven melanoma , while Tt cancer , adult Tt cancer , and MDSt cancer . The TETed in ET  and MDS ed in ET . Two othlymphoma , CML, anlymphoma  at a VAFSF3B1 and U2AF1 were identified in this study, namely U2AF1 p.Gln157Pro  and SF3B1 p.Lys700Glu  [SF3B1 p.Lys700Glu was found in a pre-PMF sample (Sample 08) which also harboured the TET2 p.Tyr1245Cys and TET2 p.Asp1314Metfs*48 variants alongside the JAK2 V617F driver mutation. The patient was 50 years of age and presented with abnormally high platelet count (1099 × 109/L), anaemia (Hb = 8.7 g/dL), and constitutional symptoms  (Table ASXL1 p.Tyr591* stopgain variant in addition to the JAK2 V617F driver mutation. The patient was 63 years of age and presented with severe anaemia (Hb = 7.3 g/dL), leucopenia (WBC = 5.5 × 109/L) and thrombocytopenia (Platelet = 54 × 109/L), with constitutional symptoms (Table Two of the most common mutations in SM84677) –37. BothMPL amplicon  at < 100 × could potentially lead to false negative results. In order for the panel to be used in MPN diagnostics, optimisation of the sequencing coverage for the MPL region is critical for the detection of driver mutations in MPL. In addition, AMPL89337 , AMPL1156 (TP53 exon 4/exon 5 chr17:7578360–7578579) and AMPL90417  were found to have average depths-of-coverage of < 1000x , and are known disease markers with diagnostic, prognostic and/or therapeutic value. First, the performance of the custom NGS panel was technically validated using reference standards in two identical but independent sequencing runs. The combined analysis of variants detected by the DNA Amplicon and Pindel tools revealed that the panel achieved high sensitivity, specificity, concordance and positive predictive values. The overall good performance of the panel was further supported in its ability to detect variants with VAF values as low as 1% Fig. : Fig. S2DNMT3A exon 17 and TP53 exon 4/exon 5 should also be used to validate the performance of the custom panel.Uneven coverage or coverage bias may originate from various steps in the NGS workflow. The use of the PCR method during NGS library preparation has been found to be the primary contributor –40. In PJAK2 and CALR, and one pathogenic stopgain variant in TET2 with a VAF of 1.4% were identified. The ASXL1 p.Leu731Tyrfs*12, p.Gln1433Gln, p.Gly646Trpfs*10 and TET2 p.Tyr1245Cys variants were identified as putative novel variants, whereas reported and likely pathogenic variants were identified in ABL1, SF3B1 and U2AF1. The study findings support the notion that the co-presence of multiple variants within a single sample results in a potentially synergistic effect that promotes disease development and progression, and contributes towards higher symptom burden, poorer prognosis, higher risk of leukaemic transformation, and drug resistance [ABL1, point mutations that confer resistance against tyrosine kinase inhibitors in patients with CML have been discovered in more than 50 different hotspots in the kinase domain [ABL1 p.Asn350Ser identified in this study may confer drug resistance in a similar fashion to those that have previously been identified.From the screening of clinical MPN samples with the custom NGS panel, 21 unique variants including MPN driver mutations in sistance –46. In Ae domain –50. It iAs MPNs are a clonally heterogenous and multifactorial group of diseases, differences in gene dosage and clonal architecture, the order of mutation acquisition and even germline predisposition can contribute towards MPN pathogenesis as well as differences in disease phenotype and prognosis , 52. In TET2 p.Tyr1631*, JAK2 V617F and U2AF1 p.Gln157Pro where the allelic frequencies were < 15%, whereas DMNT3A p.Pro385Pro was not confirmed due to nonspecific primer binding during the PCR step , monitoring of minimal residual disease, determination of disease predisposition, as well as accurate prediction of treatment response/outcome , 64. A lIn summary, the custom NGS panel enabled the detection of known MPN-associated genetic variants, as well as the identification of novel variants of potential biological significance, indicating its potential clinical utility in the genetic profiling of MPN patients. However, further performance optimisation is required especially for regions with poor coverage depth to ensure that the custom NGS panel will serve as a robust and reliable tool for the personalised management of MPNs. It is hoped that the data generated from the screening of MPN patients using the custom NGS panel will also contribute towards the MPN knowledgebase, and support the adoption of more accurate genomics-based disease classification as well as prognostic frameworks for the management of MPNs.Additional file 1.Table S1. Targeted exons of 22 MPN-associated genes in the custom NGS panel.Additional file 2.Table S2. Amplicon regions.Additional file 3.Table S3. Details of variants present in reference standards.Additional file 4.Table S4. Primers used in variant confirmation.Additional file 5.Fig. S1. Representative image of (a) Labchip gel and (b) multiple overlay electropherogram of the constructed libraries of the reference standards. The image shows the quality and quantity of input DNA for the second NGS run for the technical validation of the custom NGS panel. The majority of the constructed libraries were within the targeted size range of 400 bp.Additional file 6.Table S5. Sequencing metrics of NGS runs for the technical validation of the custom NGS panel.Additional file 7.Table S6. Amplicon coverage data for the technical validation of the custom NGS panel.Additional file 8.Fig. S2. Amplicon coverage across reference standards in the technical validation of the custom NGS panel. More details on the amplicon regions can be found in Additional file Additional file 9.Fig. S3. Gel electrophoresis images.Additional file 10. Amplicon coverage. Sheet 1 shows the genomic sequences covered by each amplicon, whereas Sheet 2 shows the depth-of-coverage per amplicon for each of the samples across the two NGS runs.Additional file 11.Fig. S4. Distribution of GC content across AMPL117202 .


By in vitro studies, we demonstrated that Integra® and PELNAC® possess superior pro-adhesive and pro-angiogenetic properties. Animal models allowed us to demonstrate the ability of preseeded ADMECs on Integra® to promote the engraftment, integration and vascularization of ADMs at the site of application.The loss of skin integrity has always represented a major challenge for clinicians dealing with dermal defects, such as ulcers , postoncologic resections  or dermatologic disorders. The introduction in recent decades of acellular dermal matrices (ADMs) supporting the repair and restoration of skin functionality represented a significant step toward achieving clean wound repair before performing skin grafts. Hard-to-heal ulcers generally depend on local ischemia and nonadequate vascularization. In this context, one possible innovative approach could be the prevascularization of matrices with vessel-forming cells (inosculation). This paper presents a comparative analysis of the most widely used dermal templates, i.e., Integra The loss of skin integrity has always represented a major challenge for clinicians, especially when dealing with fragile patients. Burns, ulcers , postoncologic resections  and dermatologic disorders  are among the most common causes of skin integrity defects ,2,3; theThe treatment of skin lesions can be either conservative or surgical, depending on the size and type of wound, as well as on patient characteristics and comorbidities. Generally, when dealing with vulnerable patients, the decision to perform surgery rather than applying conservative treatment is not always straightforward, since patient overall health status has a preponderant influence on the healing process. In fact, several local and systemic factors , pathologies and drugs can interfere with the curative process . For insIn addition, due to our ageing society and associated chronic diseases, the incidence of ulcers is steadily increasing, causing significant impairment of patient quality of life and having a major economic impact on the health care system ,14,15.The introduction in recent decades of acellular dermal matrices (ADMs) as skin substitutes represented major progress in providing coverage and support to repairing and restoring skin functionality . Since AThe use of commercial biocompatible scaffolds or dermal substitutes in regenerative medicine has been widely investigated, and several research groups are currently developing new biomaterials. One of the main applications of these matrices is the treatment of chronic wounds, ulcers or burns, in an attempt to mimic the mechanical properties of physiological skin and avoid immune rejections or toxicity .A pivotal factor for the wound healing process is graft revascularization. In fact, hard-to-heal ulcers generally depend on local ischemia, due to the formation of an impaired capillary network, with consequent low oxygenation levels and poor nutrient supply . If a maThe angiogenetic process plays a fundamental role in physiological events, such as development, wound repair and reproduction, but also in several pathological statuses, such as tumor progression. Usually, endothelial cells (ECs) remain in a quiescent state, but during angiogenesis, they can rapidly proliferate, migrate and organize themselves into tubular structures. Vascular endothelial growth factor-A (VEGF-A) plays a major role in the process via its specific receptor VEGFR-2, also known as Flk-1 or KDR, with contributions from angiopoietin-1 (ANGPT-1), integrins and several chemokines ,23,24. T® Bilayer Matrix Wound Dressing, PELNAC®, PriMatrix® Dermal Repair Scaffold, Endoform® Natural Dermal Template, and Myriad Matrix® ; mouse monoclonal antibody antihuman CD31 (#M0823 Dako), goat monoclonal antibody antimouse CD31 . All chemicals were purchased by Sigma Aldrich.The following antibodies were used: rabbit mAb antiplatelet EC adhesion molecule (PECAM)-1/CD31, mouse mAb anti-CD146, mouse mAb antivimentin, and rabbit antihuman Ki-67 purchased from Sigma Aldrich ; mouse mAb anti-von Willebrand Factor (vWF), goat antimouse fluorescein isothiocyanate (FITC)-conjugated F(ab)’, rabbit mAb anticytokeratin (CK)8/18, mouse mAb anti-Ki-67, and mouse mAb antihuman CD31 from Dako ; mouse mAb antipodoplanin was bought from Abcam ; mouse mAb antivascular endothelial growth factor receptor (VEGFR)-3 from Merk ; mouse mAb antivascular endothelial (VE)-cadherin obtained through the courtesy of E. Dejana ; mouse mAb anti-CD105 FITC-conjugated from ImmunoTools ; rabbit antihuman vimentin was bought from Santa Cruz Biotechnologies ; FITC-conjugated goat antirabbit and cyanin 3 (Cy3)-conjugated goat antirabbit were purchased from Jackson ImmunoResearch ; goat mAb antimouse PECAM-1/CD31 from R&D Systems , and antimouse IRdyev/v fetal bovine serum , 10% v/v human serum (Sigma Aldrich) and 1:100 hydrocortisone (Sigma Aldrich). ECs were positively selected with Dynabeads® CD31-conjugated magnetic beads . ADMECs were maintained at 37 °C in a humidified atmosphere in a 5% v/v CO2 incubator.Human umbilical vein ECs (HUVECs) were isolated following the protocol described by Jeffe et al. . Pregnan2). Confluent cells were fixed with 3% paraformaldehyde (PFA) for 15 min in the dark. In order to perform quenching, blocking and permeabilization, cells were then incubated with a solution of 1% bovine serum albumin (BSA), 0.1 % Triton X-100 and 50 mM glycine in Dulbecco’s Phosphate Saline Buffer  for 30 min at room temperature (RT). Primary antibodies diluted in dPBS-2% BSA and 0.7 mM CaCl2 and 0.7 mM MgCl2+ were added for 1 h at RT; secondary antibodies were incubated for 30 min at RT. Nuclei were stained with DAPI. Glass slides were mounted with a fluorescence mounting medium (Dako), and images were acquired with a Leica DM3000 microscope  using a Leica DFC320 digital camera (Leica).Cells were seeded onto round glass coverslips of 10 mm diameter previously coated with fibronectin-gelatin  following the manufacturer’s protocol, and retrotranscribed to cDNA with the SuperMix kit  as previously described [18S, GAPDH, and TBP as housekeeping genes [VEGFA, PGF, ANGPT1, KDR, FLT1, TEK, IL6, IL8/CXCL8, TNF, MCP1/CCL2, MMP2, and MMP9. Primer sequences are reported in ADMECs were seeded onto different matrices previously cut into small pieces with a biopsy punch under sterile conditions. Cells were cultured for 36 h at 37 °C in a humidified atmosphere in a 5% escribed . The expng genes . The expThe reaction was performed using the Rotor-Gene 6000 , using SYBR™ Green PCR Master Mix , following a program of 45 cycles of denaturation (60 s at 95 °C), annealing  and amplification (60 s at 72 °C).5 cells/matrix. After 5, 15 or 30 min of adhesion at 37 °C in a 5% CO2 v/v incubator, nonadherent cells were washed away with dPBS supplemented with 0.7 mM CaCl2 and 0.7 mM MgCl2. To analyze cell viability, 3--2,5-diphenyltetrazolium bromide (MTT) was added and incubated at 37 °C for 2–4 h. The purple formazan crystals were solubilized by adding dimethyl sulfoxide (DMSO) under shaking for 20 min. Absorbance was read at 570 nm using a plate reading spectrophotometer.Matrices were cut into small pieces (8 mm in diameter) using a biopsy punch under sterile conditions. ADMECs were seeded onto the matrices at a concentration of 2.5 × 10v/v CO2 incubator, 2.5 × 105 cells were resuspended in HESFM supplemented with 10% FBS and 1:100 hydrocortisone and seeded onto the matrices. After 5, 15 or 30 min of adhesion at 37 °C in a 5% v/v CO2 incubator, nonadherent cells were washed away with dPBS supplemented with 0.7 mM CaCl2 and 0.7 mM MgCl2. Cells were lysed and the plate was immediately read with Infinite200 .Matrices were cut into small pieces (8 mm in diameter) using a biopsy punch under sterile conditions. Cells were stained with 10 μg/mL of fluorescent dye FAST DiI  diluted in dPBS. After 15 min of incubation at 37 °C in a 5% v/v CO2. After 4 days, matrices were labelled with 10 μg/mL of FAST DiI diluted in dPBS and lysing buffer was added. The fluorescence was read with Infinite200 .ADMECs were cultured for 4 days in an incubator at 37 °C, 5% v/v CO2. Matrices were fixed with 3% PFA for 20 min at RT under shaking in the darkness. Matrices were incubated for 40 min at RT under shaking with primary antibodies anti-Ki-67 and antivimentin diluted in saponin A, composed of 60 mg Saponin  in 10 mL dPBS; and for 30 min at RT in darkness under shaking with LI-COR Biosciences-specific secondary antibodies. Images were acquired using Odissey CLx  and analyzed with LI-COR Image Studio Acquisition software .Matrixes were cut into small pieces (8 mm in diameter) with a biopsy punch under sterile conditions. ADMECs were seeded onto the matrices and cultured for 4 days in an incubator at 37 °C, 5% To chemically mimic hypoxia and diabetes, cells were cultured for 4 days in the presence of 100 μM deferoxamine (Sigma Aldrich) or 20 μM advanced glycation endproduct-BSA (AGE-BSA), kindly donated by Dr. Cristina Bellarosa . The concentration of deferoxamine was selected based on a previous work by Sánchez-Elsner et al. . The conv/v CO2 incubator; the supernatant was then collected. The levels of placental growth factor (PlGF), VEGF-A, TNF-α, and IL-8 were determined with a commercial ELISA kit following the manufacturer’s protocol .ADMECs were seeded onto different matrices previously cut into small pieces with a biopsy punch under sterile conditions, or grown to confluence in 24-well plates  in serum-free medium and stimulated for 24 h with interferon (IFN)-γ (100 U/mL), tumor necrosis factor (TNF)-α (100 ng/mL) or interleukin (IL)-1β (10 ng/mL) . Cells seeded onto matrices were cultured for 36 h at 37 °C in a humidified atmosphere in a 5% The initial quantitative determination of IL-8/CXCL8, monocyte chemoattractant protein (MCP)-1/CCL2, macrophage inflammatory protein (MIP)-1α/CCL3, regulated upon activation, normal T cell expressed and secreted (RANTES)/CCL5, and IL-6 was performed by a bead-based multiplex immunoassay (Biorad) and a Bioplex 200 system , as previously described .5 ECs were resuspended in HESFM and seeded for 30 min at 37 °C, 5% v/v CO2, Integra® Dermal Regeneration Template without a silicone layer (Integra® Life Science Corporation) or PELNAC® Dermal Substitute , that had previously been cut into small pieces (6 mm × 5 mm) using a sterile scalpel. After cell seeding, the scaffolds were directly implanted on the quadriceps of immunodeficient NOD scid gamma (NSG) mice [First, 2.5 × 10SG) mice . Surgica®, PELNAC® and the surrounding tissue on the quadricep were collected, transversely cut in half and fixed overnight using 2% v/v PFA  in dPBS 1X solution (Santa Cruz). The following day, PFA solution was removed and replaced with 20% sucrose cryoprotector solution . The samples were cut into 8-μm sections using cryostat. The sections were thawed and dried at room temperature for 5 min. After a wash in 1X dPBS, samples were permeabilized using 0.5% v/v Triton X-100 in 1X dPBS for 15 min. The blocking step was performed using 5% w/v in 1X dPBS solution for 1 h. The sections were incubated overnight at 4 °C using a humid chamber with the primary antibody, diluted at a ratio of 1:200 v/v with 1% v/v BSA, 0.1% v/v Tween-20 in 1X dPBS solution. The next day, the slides were washed once in 0.02% v/v Tween 20 in 1X dPBS solution for 5 min and twice in 1X dPBS for 5 min each. The incubation with secondary antibody was performed for 2 h at RT in a humid chamber diluting the secondary antibody 1:500 v/v with 1% v/v BSA, 0,1% v/v Tween-20 in 1X dPBS solution. The tissues were washed once in 0.02% v/v Tween 20 in 1X dPBS solution for 5 min and twice in 1X dPBS for 5 min each. Nuclei were counterstained with Hoechst 33342 (#H3570 Invitrogen) diluted 1:5000 in 1X dPBS for 7 min. Subsequently, slides were washed three times with 1X dPBS and mounted using Mowiol mounting medium (Sigma-Aldrich).The mice were sacrificed by cervical dislocation after anesthesia with 5% isoflurane. IntegraADMECs were isolated from skin biopsies of adult patients undergoing reductive plastic surgery. We carried out an extensive characterization by immunofluorescence (IF) A in orde®, PELNAC®, PriMatrix®, Endoform®, or Myriad®). After 5, 15 or 30 min of incubation, the scaffolds were extensively washed to remove unattached cells, and adherent ADMECs were labelled with the viability marker MTT in a 24-well plate , a fluorescence scanner which is able to detect multiple layers on the z-axis. As reported in ® and PELNAC® exceeded a proliferation percentage of 40%, whereas those on PriMatrix®, Endoform® and Myriad® showed a lower proliferation index.In order to evaluate the proliferative boost induced by the different dermal substitutes, ADMECs were grown onto the scaffolds for 36 h and then labelled with FAST DiI. As shown in ®, which manifested a statistically lower proliferative rate in both conditions. Even if not significantly, Integra® and Myriad® demonstrated the best performance under hypoxic and diabetic conditions.With the aim of mimicking a chronic wound microenvironment, we cultured ADMECs under hypoxic conditions  or diabetogenic conditions . The results shown in ®, Endoform®, and considering the great variability of the results obtained with Myriad®, subsequent studies were conducted only on Integra® and PELNAC®. To assess the mRNA expression of angiogenic-promoting factors , receptors  and tissue remodeling factors (MMP2 and MMP9) expressed by ADMECs cultured on Integra® and PELNAC®, we performed RT-qPCR after seeding the cells onto the scaffolds for 36 h.Having demonstrated the poor pro-adhesive and proproliferative properties of PriMatrix® induced the activation of almost all angiogenetic factor genes compared to 2D cell culture (except for TEK and MMP2). PELNAC® was able to activate numerous angiogenic genes and, in some cases, in an even stronger manner than Integra®   A–H.Moreover, the upregulation of soluble angiogenetic factors  was also confirmed in terms of protein secretion I–K.restitutio ad integrum of wounds [® was able to upregulate the expression of the healing-promoting cytokines as follows: a two-fold increase was observed in the expression of TNF, a 2.5-fold increase in the upregulation of IL8/CXCL8 was observed; a seven-fold increase of MCP1/CCL2 was observed; and a two-fold of IL6 was observed, as compared to cells seeded without scaffolds. On the other hand, PELNAC® yielded only a two-fold upregulation of IL8.Since the formation of granulation tissue and leukocyte recruitment is fundamental for the f wounds , we wantThe cytokine upregulation observed for gene expression was not confirmed at the protein level. We confirmed a slight, nonsignificant upregulation of IL-8/CXCL8 , but we ® or PELNAC® for 30 min and subcutaneously implanted on the quadriceps of NSG recipient mice. Histological analysis at 10 days revealed the presence of CD31-stained human ECs that colonized Integra®  quantification of the total number of living cells present in the matrix after 36 h, and (ii) the percentage of proliferating cells (positive for Ki-67) normalized for the total number of cells (positive for vimentin). The results confirmed those obtained for the adhesion experiments, indicating that Integra®, PELNAC®, and Myriad® were the most promising dermal substitutes in terms of local proliferation of vascular cells.The principal aim of this study was to enhance revascularization to successfully promote wound healing . Since t® were able to proliferate under the hypoxia condition. In general, hypoxia signaling is essential during tissue and organ development, but this condition is also important in specific organs in healthy adults. In fact, since the hypoxia-inducible factor(HIF)-α is not hydroxylated by prolyl hydroxylase under hypoxic conditions, it can activate the HIF-mediated transcriptional program, which also includes angiogenesis. Hypoxia triggers the transcription of angiogenic genes including VEGFA, PGF, PDGFB and ANGPT1 and 2. Furthermore, it posttranscriptionally controls pro-angiogenic chemokines and receptors, inducing the migration of progenitors to the site of angiogenesis. Hypoxia can also promote EC proliferation and sprouting in addition to matrix remodeling [db/db mice [Chronic wounds are defined as a discontinuity in the skin barrier lasting longer than 42 days. Risk factors for the delay of physiological wound healing are chronic disease, diabetes, age, vascular insufficiency, nutritional deficit and some local factors . With thmodeling . The phy/db mice .®, PELNAC® and Myriad®) were able to support at least 10% of proliferating cells in a diabetes-like condition.Nonenzymatic glycation of intra- and extra- cellular proteins by hyperglycemia may cause tissue damage. In fact, AGEs irreversibly bind to proteins. Several studies have shown that AGEs induce endothelial dysfunction. In particular, they increase oxidant stress, induce permeability, stimulate the expression of adhesion molecules and augment the migration of T cells and macrophages . For thi® and PELNAC® because they demonstrated the highest vascularization properties and low variability in the results. The angiogenic drive induced by dermal substitutes was evaluated by RT-qPCR for the main pro-angiogenic factors/receptors and tissue remodeling factors. With the exception of TEK, the ANGPT-1 receptor, both matrices (Integra® and PELNAC®) were shown to activate the expression of all genes under study. Furthermore, we also confirmed, at the protein level, the upregulation of VEGF-A synthesis by PELNAC® of and the upregulation of PlGF secretion by Integra®.We successively focused our experiments on Integracondition sine qua non to complete restitutio ad integrum. Thus, we performed RT-qPCR with the aim of evaluating the expression of healing-promoting cytokines. In this case, Integra® was shown to be able to upregulate the gene expression of TNF, IL8/CXCL8, MCP1/CCL2 and IL6. These data were partially confirmed at the protein level, since Integra® seemed to downregulate the production of TNF-α, but also to increase the production of IL-8/CXCL8. In this case, Integra® also showed the most promising results by upregulating the genes involved in leukocyte recruitment, e.g., IL-8/CXCL8 and MCP1/CCL2, although this also led to the upregulation of TNF-α and IL-6, cytokines involved in chronic inflammation.Inflammation is one of the physiological responses to wound healing . The for® to promote the engraftment, integration and vascularization of clinically employed dermal regeneration scaffolds at the site of application. In vivo experiments to evaluate the efficacy of human cell therapies require the use of immunocompromised mice (in our case NSG mice) [Finally, in vivo experiments were undertaken to confirm our in vitro observations. We assessed the ability of preseeded ADMECs on IntegraSG mice) . Given tSG mice) .®, PELNAC® and Myriad® demonstrated the best performance in terms of EC colonization and proliferation, even under hypoxic and diabetic conditions. The interaction of ECs with both Integra® and PELNAC® induced the expression of multiple angiogenetic factors. Integra® was the most effective scaffold in promoting EC engraftment and vascularization in vivo. Thus, the implantation of dermal ECs in combination with ECMs represents a promising strategy for tissue revascularization. Integra® remains the “gold standard” ADM for this application.In conclusion, Integra