Targeted siRNA lipid nanoparticles for the treatment of KRAS-mutant tumors

K-RAS is a highly relevant oncogene that is mutated in approximately 90% of pancreatic cancers and 20–25% of lung adenocarcinomas. The aim of this work was to develop a new anti-KRAS siRNA therapeutic strategy through the engineering of functionalized lipid nanoparticles (LNPs). To do this, first, a potent pan anti-KRAS siRNA sequence was chosen from the literature and different chemical modifications of siRNA were tested for their transfection efficacy (KRAS knockdown) and anti-proliferative effects on various cancer cell lines. Second, a selected siRNA candidate was loaded into tLyp-1 targeted and non-targeted lipid nanoparticles (LNPs). The biodistribution and antitumoral efficacy of selected siRNA-loaded LNP-prototypes were evaluated in vivo using a pancreatic cancer murine model (subcutaneous xenograft CFPAC-1 tumors). Our results show that tLyp-1-tagged targeted LNPs have an enhanced accumulation in the tumor compared to non-targeted LNPs. Moreover, a significant reduction in the pancreatic tumor growth was observed when the anti-KRAS siRNA treatment was combined with a classical chemotherapeutic agent, gemcitabine. In conclusion, our work demonstrates the benefits of using a targeting approach to improve tumor accumulation of siRNA-LNPs and its positive impact on tumor reductionThis work was supported by the 2-INTRATARGET project (PCIN-2017-129/AEI) funded by MINECO-PCIN-2017-129/AEI, under the frame of EuroNanoMed III; by Consellería de Educación e Ordenación Universitaria, Xunta de Galicia's Grupos de referencia competitiva (grant number ED431C 2017/09). The authors thank TÜBİTAK (The Scientific and Technical Research Council of Turkey) for supporting this project (Project number : 217S068). S.A acknowledges the financial support for his postdoctoral research by the 2-INTRATARGET project (PCIN-2017-129/AEI) funded by MINECO-PCIN-2017-129/AEI, under the frame of EuroNanoMed IIIS

Functional Exhaustion Of Cd4(+) T Cells Induced By Co-Stimulatory Signals From Myeloid Leukaemia Cells

To cope with immune responses, tumour cells implement elaborate strategies such as adaptive resistance and induction of T-cell exhaustion. T-cell exhaustion has been identified as a state of hyporesponsiveness that arises under continuous antigenic stimulus. Nevertheless, contribution of co-stimulatory molecules to T-cell exhaustion in cancer remains to be better defined. This study explores the role of myeloid leukaemia-derived co-stimulatory signals on CD4(+) T helper (Th) cell exhaustion, which may limit anti-tumour immunity. Here, CD86 and inducible T-cell co-stimulator ligand (ICOS-LG) co-stimulatory molecules that are found on myeloid leukaemia cells supported Th cell activation and proliferation. However, under continuous stimulation, T cells co-cultured with leukaemia cells, but not with peripheral blood monocytes, became functionally exhausted. These in vitro-generated exhausted Th cells were defined by up-regulation of programmed cell death 1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), lymphocyte activation gene 3 (LAG3) and T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) inhibitory receptors. They were reluctant to proliferate upon re-stimulation and produced reduced amounts of interleukin-2 (IL-2), tumour necrosis factor- (TNF-) and interferon- (IFN-). Nonetheless, IL-2 supplementation restored the proliferation capacity of the exhausted Th cells. When the co-stimulation supplied by the myeloid leukaemia cells were blocked, the amount of exhausted Th cells was significantly decreased. Moreover, in the bone marrow aspirates from patients with acute myeloid leukaemia (AML) or myelodysplastic syndrome (MDS), a subpopulation of Th cells expressing PD-1, TIM-3 and/or LAG3 was identified together with CD86(+) and/or ICOS-LG(+) myeloid blasts. Collectively, co-stimulatory signals derived from myeloid leukaemia cells possess the capacity to facilitate functional exhaustion in Th cells.Wo

Folic acid decoration of mesoporous silica nanoparticles to increase cellular uptake and cytotoxic activity of doxorubicin in human breast cancer cells

© 2021 Elsevier B.V.Breast cancer is the most frequent cancer among women and impacts over two million women each year. Although many different types of anticancer agents are available for breast cancer treatment, doxorubicin is one of the most widely used drug. However, doxorubicin related side effects such as heart failure and arrhythmia limit its usage. To overcome this limitation and improve doxorubicin effectiveness, pegylated liposomal doxorubicin formulation Doxil®/Caelyx® was developed. Although cardiotoxicity related side effects were reduced with liposomal doxorubicin formulations, a superior effect was not obtained and better approaches are still needed. In this study, it was aimed to develop a more effective doxorubicin formulation than Doxil® and to evaluate its anticancer activity. In order to achieve this goal, small sized mesoporous silica nanoparticles (MSNs) (~50 nm) were obtained, actively targeted with folic acid conjugation and loaded with doxorubicin. The obtained nanoparticles were fully characterized, conjugation was verified, and pH dependent drug release profile was shown. The nanoparticles’ anticancer activity was investigated in detail on the ZR-75-1 and T47-D breast cancer cell lines. Fluorescence microscope and flow cytometry studies revealed that the cellular uptake of doxorubicin could be enhanced with small sized MSNs. Moreover, folic acid conjugation made a tangible contribution to this effect. Additionally, similar results were also obtained in cytotoxicity studies on both cell lines. In conclusion, actively targeted small sized MSNs may be a promising approach to potentiate the anticancer effect of doxorubicin

Myeloid maturation potentiates STAT3-mediated atypical IFN-γ signaling and upregulation of PD-1 ligands in AML and MDS.

Interferon (IFN)-γ is the major mediator of anti-tumor immune responses; nevertheless, cancer cells use intrigue strategies to alter IFN-γ signaling and avoid elimination. Understanding the immune regulatory mechanisms employed by acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cells upon exposure to IFN-γ is critical for development of immunotherapy and checkpoint blockade therapy approaches. This study aims to explore the influence of myeloid maturation on IFN-γ-induced PD-L1 and PD-L2 expression and on pro-leukemogenic transcription factor STAT3 signaling in AML and MDS. Stimulation of myeloid blasts' maturation by all-trans retinoic acid (ATRA) or 1α,25-dihydroxyvitamin D3 (vitamin D) increased the CD11b fraction that expressed PD-1 ligands in response to IFN-γ. Intriguingly, STAT3 pathway was potently induced by IFN-γ and strengthened upon prolonged exposure. Nonetheless, STAT3-mediated atypical IFN-γ signaling appeared as a negligible factor for PD-L1 and PD-L2 expression. These negative influences of IFN-γ could be alleviated by a small-molecule inhibitor of STAT3, stattic, which also inhibited the upregulation of PD-L1. In conclusion, induction of myeloid maturation enhances the responsiveness of AML and MDS cells to IFN-γ. However, these malignant myeloid cells can exploit both STAT3 pathway and PD-1 ligands to survive IFN-γ-mediated immunity and maintain secondary immune resistance

Immune checkpoint status and exhaustion‐related phenotypes of CD8+ T cells from the tumor‐draining regional lymph nodes in breast cancer

Abstract Background Functional status of T cells determines the responsiveness of cancer patients to immunotherapeutic interventions. Even though T cell‐mediated immunity is inaugurated in the tumor‐adjacent lymph nodes, peripheral blood has been routinely sampled for testing the immunological assays. The purpose of this study is to determine the immune checkpoint molecule expression and the exhaustion‐related phenotype of cytotoxic T cells in the regional lymph nodes from breast cancer patients. Patients and methods Multicolor immunophenotyping was used to determine the expression of PD‐1, TIM‐3, LAG3, CTLA‐4, CCR7, CD45RO, CD127, CD25, CXCR5, and ICOS molecules on CD3+CD4−CD56−CD8+ cytotoxic T cells freshly obtained from the lymph nodes and the peripheral blood samples of the breast cancer patients. The results were assessed together with the clinical data. Results A population of cytotoxic T cells was noted with high PD‐1 and CXCR5 expression in the lymph nodes of the breast cancer patients. Co‐expression of PD‐1, CXCR5, TIM‐3, and ICOS indicated a follicular helper T cell (Tfh)‐like, exhaustion‐related immunophenotype in these cytotoxic T cells. Only a minor population with CTLA‐4 and LAG3 expression was noted. The PD‐1+CXCR5+ cytotoxic T cells largely displayed CD45RO+CCR7+ central memory markers. The amount of CXCR5‐expressing PD‐1− cytotoxic T cells was elevated in the lymph nodes of the patients. Conclusion The regional lymph nodes of breast cancer patients harbor Tfh‐like exhausted cytotoxic T lymphocytes with high PD‐1 and TIM‐3 checkpoint molecule expression. The immunological conditions in the regional lymph nodes should be implicated for immune checkpoint immunotherapy (ICI) of cancer

Design and Synthesis of Novel Peptidomimetics for Cancer Immunotherapy

Tumor cells benefit from some certain signals, which are referred to as “immune checkpoints”, to escape immune-mediated destruction. With that in mind, it is believed that the blockade of these points, such as programmed cell death Ligand-1 (PD-L1) and programmed cell death 1 (PD-1), can restore an adaptative immune response against tumoral cells. In this study, we have designed and synthesized some novel peptidomimetics with a 2-aminobenzathiazole scaffold, which targets the PD-1/PDL-1 pathway. In the viability assay, it was found that these compounds decreased the proliferation of peripheral blood mononuclear cells in the concentration of 10 uM. Overall, our results indicate that these novel compounds are potential checkpoint inhibitors for cancer immunotherapy

Serial immunological parameters in a phase II trial of exemestane and low-dose oral cyclophosphamide in advanced hormone receptor-positive breast cancer.

BACKGROUND AND PURPOSE: Resistance to endocrine therapies in hormone receptor (HR)-positive breast cancer is a significant challenge. Prior studies have shown that low-dose oral cyclophosphamide can transiently deplete regulatory T cells (Tregs) and improve anti-tumor immunity. We investigated the combination of exemestane with cyclophosphamide in patients with advanced HR-positive breast cancer and assessed changes in circulating immune cell subsets. METHODS: This was a single-arm phase II trial of exemestane with cyclophosphamide in patients with metastatic HR-positive/HER2-negative breast cancer who had progressed on prior endocrine therapy (ClinicalTrials.gov: NCT01963481). Primary endpoint was progression-free survival (PFS) at 3 months (RECIST 1.1). Secondary objectives included median PFS, objective response rate, duration of response, and safety. Circulating Tregs (FOXP3 RESULTS: Twenty-three patients were enrolled. Treatment was well tolerated, without grade 4/5 toxicities. Objective responses were seen in 6/23 patients (26.1%; 95% CI 10.2-48.4%) and were durable (median 11.6 months). Three-month PFS rate was 50.1% (95% CI 33.0-76.0%); median PFS was 4.23 months (95% CI 2.8-11.7). No treatment-related decrease in Tregs was observed. However, elevated baseline levels of Naïve Tregs [greater than 2.5 (the median of the naïve Tregs)] were associated with relative risk of disease progression or death [hazard ratio 11.46 (95% CI 2.32-56.5)]. In addition, the baseline levels of Naïve Tregs (adj-p = 0.04), Memory Tregs (adj-p = 0.003), CD4 + Central Memory T cells (adj-p = 0.0004), PD-1 + CD4 + Central Memory T cells (adj-p = 0.008), and PD-1 + CD4 + Effector Memory T cells (adj-p = 0.009) were significantly greater in the patients than in the healthy controls; the baseline levels of %CD4 + Naïve T cells (adj-p = 0.0004) were significantly lower in patients compared with healthy controls (n = 40). CONCLUSION: Treg depletion was not observed with low-dose cyclophosphamide when assessed by the specific marker FOXP3 + Helios +; however, baseline naïve Tregs were associated with 3-month PFS. Exemestane/cyclophosphamide combination had favorable safety profile with evidence of clinical activity in heavily pretreated patients. Breast Cancer Res Treat 2018 Feb; 168(1):57-67