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The brain is one of the most common metastatic sites among breast cancer patients, especially in those who have Her2-positive or triple-negative tumors. The brain microenvironment has been considered immune privileged, and the exact mechanisms of how immune cells in the brain microenvironment contribute to brain metastasis remain elusive. In this study, we found that neutrophils are recruited and influenced by c-Met high brain metastatic cells in the metastatic sites, and depletion of neutrophils significantly suppressed brain metastasis in animal models. Overexpression of c-Met in tumor cells enhances the secretion of a group of cytokines, including CXCL1/2, G-CSF, and GM-CSF, which play critical roles in neutrophil attraction, granulopoiesis, and homeostasis. Meanwhile, our transcriptomic analysis demonstrated that conditioned media from c-Met high cells significantly induced the secretion of lipocalin 2 (LCN2) from neutrophils, which in turn promotes the self-renewal of cancer stem cells. Our study unveiled the molecular and pathogenic mechanisms of how crosstalk between innate immune cells and tumor cells facilitates tumor progression in the brain, which provides novel therapeutic targets for treating brain metastasis.

Philadelphia chromosome positive B cell acute lymphoblastic leukemia (Ph+ ALL) is an aggressive cancer of the bone marrow. The addition of tyrosine kinase inhibitors (TKIs) has improved outcomes but many patients still suffer relapse and novel therapeutic agents are needed. KPC34 is an orally available, novel phospholipid conjugate of gemcitabine, rationally designed to overcome multiple mechanisms of resistance, inhibit the classical and novel isoforms of protein kinase C, is able to cross the blood brain barrier and is orally bioavailable. KPC34 had an IC50 in the nanomolar range against multiple ALL cell lines tested but was lowest for Ph+ lines. In mice bearing either naïve or resistant Ph+ ALL, KPC34 treatment resulted in significantly improved survival compared to cytarabine and gemcitabine. Treatment with KPC34 and doxorubicin was more effective than doxorubicin and cytarabine. Mice with recurrence of their ALL after initial treatment with cytarabine and doxorubicin saw dramatic improvements in hind limb paralysis after treatment with KPC34 demonstrating activity against established CNS disease. Consistent with this KPC34 was better than gemcitabine at reducing CNS leukemic burden. These promising pre-clinical results justify the continued development of KPC34 for the treatment of Ph+ALL.

Malignant pleural effusion (MPE) is indicative of terminal malignancy with a uniformly fatal prognosis. Often, two distinct compartments of tumour microenvironment, the effusion and disseminated pleural tumours, co-exist in the pleural cavity, presenting a major challenge for therapeutic interventions and drug delivery. Clinical evidence suggests that MPE comprises abundant tumour-associated myeloid cells with the tumour-promoting phenotype, impairing antitumour immunity. Here we developed a liposomal nanoparticle loaded with cyclic dinucleotide (LNP-CDN) for targeted activation of stimulators of interferon genes signalling in macrophages and dendritic cells and showed that, on intrapleural administration, they induce drastic changes in the transcriptional landscape in MPE, mitigating the immune cold MPE in both effusion and pleural tumours. Moreover, combination immunotherapy with blockade of programmed death ligand 1 potently reduced MPE volume and inhibited tumour growth not only in the pleural cavity but also in the lung parenchyma, conferring significantly prolonged survival of MPE-bearing mice. Furthermore, the LNP-CDN-induced immunological effects were also observed with clinical MPE samples, suggesting the potential of intrapleural LNP-CDN for clinical MPE immunotherapy. Malignant pleural effusion (MPE) is the terminal stage of cancer and the current standard of care for MPE is largely palliative. Here the authors design a liposomal nanoparticle loaded with cyclic dinucleotide for targeted activation of STING signalling in macrophages and dendritic cells and show that, on intrapleural administration, the nanoparticle effectively mitigates the immune cold MPE and significantly augments the checkpoint blockade immunotherapy in a mouse MPE model and clinical patients’ samples.

We have previously synthesized a phospholipid-gemcitabine conjugate and a phospholipid-cytosine arabinoside conjugate that we tested in different human cancer cell lines. The gemcitabine conjugate was more cytotoxic to the cancer cells tested than the cytosine arabinoside (ara-C) conjugate. The focus here was to elucidate the mechanism of action of the conjugate molecule and its ability to bypass certain drug-resistance mechanisms. In contrast to gemcitabine, the gemcitabine conjugate did not enter the cell via the human equilibrative nucleoside transporter (hENT1). Additionally, the gemcitabine conjugate was not a substrate for the multidrug resistance efflux pump, MDR-1, even though the molecule is more lipophilic. Finally, we showed that deoxycytidine kinase (dCK) was not required for the activation of the gemcitabine conjugate. As expected, cells overexpressing dCK were more sensitive to gemcitabine whereas cells overexpressing dCK were not more sensitive to the gemcitabine conjugate. Taken together, these results suggest that the gemcitabine conjugate may be therapeutically superior to gemcitabine due to the conjugate's ability to bypass three resistance mechanisms that often render gemcitabine ineffective as an anticancer agent.

Phospholipid nucleoside conjugates and nucleosides with chemical additions to the hydroxyl and amino moieties have been used since the 1970s in order to increase the biological activity of the parent compound. Previous investigators have found that adding lipid moieties to ara-C or chemically linking ara-C to a phospholipid creates a prodrug that exhibits superior cytotoxic activity compared to ara-C alone when used in animal tumor models. The novel ara-C molecules reveal different pharmacological profiles from the parent compound such as decreased catabolism by cytidine deaminase, increased plasma half-life, and release of nucleoside monophosphate, a reaction that bypasses the rate limiting initial nucleoside phosphorylation. Additionally, these compounds were able to penetrate the blood-brain barrier and were active against tumor cells implanted i.c. The purpose of this review is to briefly cover the history and successes of previous investigators who have synthesized and tested these phospholipid ara-C conjugates, to discuss recent phospholipid ara-C and fludarabine conjugates, and to discuss the synthetic design and synthesis of a novel phospholipid gemcitabine conjugate. These phospholipid nucleoside conjugates possess the potential to have superior anti-neoplastic cytotoxicity profiles with fewer side effects than the parent nucleoside and merit further investigation.

Cytosine arabinoside induces apoptosis and this cell death process is influenced by protein kinase C signaling events in leukemic cells. We present findings that extend these observations to include another deoxycytidine analog, gemcitabine, which is more potent in solid tumors. Gemcitabine induced programmed cell death in BG-1 human ovarian cancer cells based on biochemical and morphologic analyses. The DNA was fragmented in BG-1 cells exposed to gemcitabine (0.5 microM, 1.0 microM and 10 microM) for 8 h, but gemcitabine treatment did not induce internucleosomal DNA degradation. Scanning and transmission electron microscopy of BG-1 cells showed morphologic changes associated with apoptosis in response to gemcitabine: membrane blebbing, the formation of apoptotic bodies and chromatin condensation. Thus, BG-1 cells undergo programmed cell death in response to gemcitabine treatment without internucleosomal DNA fragmentation. Furthermore, gemcitabine (10 microM) activated protein kinase C in BG-1 cells and the phosphorylation of the endogenous protein kinase C substrate, myristoylated alanine-rich C kinase substrate, was increased following exposure of BG-1 cells to gemcitabine for up to 6 h. Clonogenicity studies with gemcitabine in combination with various protein kinase C-modulating agents demonstrated that gemcitabine cytotoxicity was influenced by protein kinase C signaling events in BG-1 cells. Short-term (1 h) exposure to TPA (1 or 10 nM) followed by gemcitabine (0.5 microM for 4 h) did not alter the response to gemcitabine. However, a 24-h exposure to TPA followed by gemcitabine resulted in synergistic cytotoxicity, while coincubation of TPA with a PKC inhibitor (e.g. bisindolylmaleimide or calphostin-C) in this regimen abrogated the synergistic response. Based on our findings, it is plausible that gemcitabine therapy could be improved by modulating PKC signaling events linked to drug-induced apoptosis/cytotoxicity.

Modern synthetic biology depends on the manufacture of large DNA constructs from libraries of genes, regulatory elements or other genetic parts. Type IIS-restriction enzyme-dependent DNA assembly methods (e.g., Golden Gate) enable rapid one-pot, ordered, multi-fragment DNA assembly, facilitating the generation of high-complexity constructs. The order of assembly of genetic parts is determined by the ligation of flanking Watson-Crick base-paired overhangs. The ligation of mismatched overhangs leads to erroneous assembly, and the need to avoid such pairings has typically been accomplished by using small sets of empirically vetted junction pairs, limiting the number of parts that can be joined in a single reaction. Here, we report the use of a comprehensive method for profiling end-joining ligation fidelity and bias to predict highly accurate sets of connections for ligation-based DNA assembly methods. This data set allows quantification of sequence-dependent ligation efficiency and identification of mismatch-prone pairings. The ligation profile accurately predicted junction fidelity in ten-fragment Golden Gate assembly reactions, and enabled efficient assembly of a lac cassette from up to 24-fragments in a single reaction. Application of the ligation fidelity profile to inform choice of junctions thus enables highly flexible assembly design, with >20 fragments in a single reaction.