The efficacy of chemotherapeutic drug combinations may be predicted by concordance of gene response to the single agents

Determining the expression of genes in response to different classes of chemotherapeutic drugs may allow for a better understanding as to which may be used effectively in combination. In the present study, the human colorectal cancer cell line HCT116 was cultured with equi‑active concentrations of a series of anti‑cancer agents. Gene expression profiles were then measured by whole‑genome microarray. Although each drug induced a unique signature of gene expression in tumour cells, there were marked similarities between certain drugs, even in those from different classes. For example, the antimalarial agent artesunate and the platinum‑containing alkylating agent, oxaliplatin, produced a very similar mRNA expression pattern in HCT116 cells with ~14,000 genes being affected by the two drugs in the same way. Furthermore, the overall correlation of gene responses between two agents could predict whether their use in combination would lead to a greater or lesser effect on cell number, determined experimentally, than predicted by single agent experiments. The results indicated that even when working through different mechanisms, combining drugs that initiate a similar transcriptional response may constitute the best option for determining drug‑combination strategies for the treatment of cancer

Gemcitabine alters the proteasome composition and immunopeptidome of tumour cells

The antigenic makeup of tumour cells can have a profound effect on the progression of cancer and success of immunotherapies. Therefore, one strategy to improve the efficacy of cancer treatments is to augment the antigens displayed by tumours. The present study explores how the recognition of tumour cells may be altered by non-cytotoxic concentrations of gemcitabine (GEM). Testing a panel of chemotherapeutics in human cancer cell lines in vitro, it was found that GEM increased surface expression of HLA-A,B,C and that underlying this were specific increases in β-2-microglobulin and immunoproteasome subunit proteins. Furthermore, the peptide antigen repertoire displayed on HLA class I was altered, revealing a number of novel antigens, many of which that were derived from proteins involved in the DNA-damage response. Changes in the nature of the peptide antigens eluted from HLA-A,B,C after GEM treatment consisted of amino acid anchor-residue modifications and changes in peptide length which rendered peptides likely to favour alternative HLA-alleles and increased their predicted immunogenicity. Signalling through the MAPK/ERK and NFκB/RelB pathways was associated with these changes. These data may explain observations made in previous in vivo studies, advise as to which antigens should be used in future vaccination protocols and reinforce the idea that chemotherapy and immunotherapy could be used in combination

The cytotoxic molecule granulysin is capable of inducing either chemotaxis or fugetaxis in dendritic cells depending on maturation: a role for Vδ2+ γδ T cells in the modulation of immune response to tumour?

Release of granulysin by γδ T cells contributes to tumour cell killing. A cytolytic 9kDa isoform of granulysin kills tumour cells directly, while a 15kDa precursor has been hypothesised to cause both the maturation and migration of dendritic cell (DC) populations. Recruiting DC to a tumour is beneficial as these cells initiate adaptive immune responses, which contribute to the eradication of malignancies. In this study, Vδ2+ γδ T cells were activated by stimulation of peripheral blood mononuclear cells (PBMC) with zoledronic acid (ZA) or Bacillus Calmette Guérin (BCG), or were isolated and cultured with tumour targets. While a large proportion of resting Vδ2+ γδ T cells expressed 15kDa granulysin, 9kDa granulysin expression was induced only after stimulation with BCG. Increased levels of activation and granulysin secretion were also observed when Vδ2+ γδ T cells were cultured with the human B cell lymphoma line Daudi. High concentrations of recombinant 15kDa granulysin caused migration and maturation of immature DC, and also initiated fugetaxis in mature DC. Conversely, low concentrations of recombinant 15kDa granulysin resulted in migration of mature DC, but not immature DC. Our data therefore support the hypothesis that Vδ2+ γδ T cells can release granulysin, which may modulate recruitment of DC, initiating adaptive immune responses

Supernatants from lymphocytes stimulated with Bacillus Calmette-Guerin can modify the antigenicity of tumours and stimulate allogeneic T-cell responses

BACKGROUND: Reduced expression of class 1 human leucocyte antigens (HLA1) is often a mechanism by which tumours evade surveillance by the host immune system. This is often associated with an immune function that is unable to mount appropriate responses against disease, which can result in a state that favours carcinogenesis. METHODS: In the current study, we have explored the effects of Bacillus Calmette-Guerin (BCG) on the cytokine output of leucocytes, which is a key determinant in generating antitumour action, and have also assessed the effect of these cytokine cocktails on HLA1 expression in solid tumour cell lines. RESULTS: BCG potently activated a broad range of leucocytes, and also enhanced the production of cytokines that were Th(1)-predominant. Supernatants from BCG-treated leucocytes significantly increased the expression of HLA1 on the surface of cancer cell lines, which correlated with increased cytolytic T-cell activity. We also showed that the increased HLA1 expression was associated with activation of intracellular signalling pathways, which was triggered by the increases in the Th(1)-cytokines interferon-γ and tumour necrosis factor-α, as counteracting their effects negated the enhancement. CONCLUSION: These studies reaffirm the role of BCG as a putative immunotherapy through their cytokine-modifying effects on leucocytes and their capacity to enhance tumour visibility

Functional LTCC-β₂AR complex needs Caveolin-3 and is disrupted in heart failure

Background: Beta-2 adrenergic receptors (β2ARs) but not beta-2 adrenergic receptors (β1ARs) form a functional complex with L-type Ca2+ channels (LTCCs) on the cardiomyocyte membrane. However, how microdomain localization in the plasma membrane affects the function of these complexes is unknown. We aim to study the coupling between LTCC and β adrenergic receptors in different cardiomyocyte microdomains, the distinct involvement of PKA and CAMKII (Ca2+/calmodulin-dependent protein kinase II) and explore how this functional complex is disrupted in heart failure. Methods: Global signaling between LTCCs and β adrenergic receptors was assessed with whole-cell current recordings and western blot analysis. Super-resolution scanning patch-clamp was used to explore the local coupling between single LTCCs and β1AR or β2AR in different membrane microdomains in control and failing cardiomyocytes. Results: LTCC open probability (Po) showed an increase from 0.054±0.003 to 0.092±0.008 when β2AR was locally stimulated in the proximity of the channel (<350 nm) in the transverse tubule microdomain. In failing cardiomyocytes, from both rodents and humans, this transverse tubule coupling between LTCC and β2AR was lost. Interestingly, local stimulation of β1AR did not elicit any change in the Po of LTCCs, indicating a lack of proximal functional interaction between the two, but we confirmed a general activation of LTCC via β1AR. By using blockers of PKA and CaMKII and a Caveolin-3-knockout mouse model, we conclude that the β2AR-LTCC regulation requires the presence of caveolin-3 and the activation of the CaMKII pathway. By contrast, at a cellular “global” level PKA plays a major role downstream β1AR and results in an increase in LTCC current. Conclusions: Regulation of the LTCC activity by proximity coupling mechanisms occurs only via β2AR, but not β1AR. This may explain how β2ARs tune the response of LTCCs to adrenergic stimulation in healthy conditions. This coupling is lost in heart failure; restoring it could improve the adrenergic response of failing cardiomyocytes

Rab3D is critical for secretory granule maturation in PC12 cells.

Neuropeptide- and hormone-containing secretory granules (SGs) are synthesized at the trans-Golgi network (TGN) as immature secretory granules (ISGs) and complete their maturation in the F-actin-rich cell cortex. This maturation process is characterized by acidification-dependent processing of cargo proteins, condensation of the SG matrix and removal of membrane and proteins not destined to mature secretory granules (MSGs). Here we addressed a potential role of Rab3 isoforms in these maturation steps by expressing their nucleotide-binding deficient mutants in PC12 cells. Our data show that the presence of Rab3D(N135I) decreases the restriction of maturing SGs to the F-actin-rich cell cortex, blocks the removal of the endoprotease furin from SGs and impedes the processing of the luminal SG protein secretogranin II. This strongly suggests that Rab3D is implicated in the subcellular localization and maturation of ISGs

Improved Endpoints for Cancer Immunotherapy Trials

Unlike chemotherapy, which acts directly on the tumor, cancer immunotherapies exert their effects on the immune system and demonstrate new kinetics that involve building a cellular immune response, followed by changes in tumor burden or patient survival. Thus, adequate design and evaluation of some immunotherapy clinical trials require a new development paradigm that includes reconsideration of established endpoints. Between 2004 and 2009, several initiatives facilitated by the Cancer Immunotherapy Consortium of the Cancer Research Institute and partner organizations systematically evaluated an immunotherapy-focused clinical development paradigm and created the principles for redefining trial endpoints. On this basis, a body of clinical and laboratory data was generated that supports three novel endpoint recommendations. First, cellular immune response assays generate highly variable results. Assay harmonization in multicenter trials may minimize variability and help to establish cellular immune response as a reproducible biomarker, thus allowing investigation of its relationship with clinical outcomes. Second, immunotherapy may induce novel patterns of antitumor response not captured by Response Evaluation Criteria in Solid Tumors or World Health Organization criteria. New immune-related response criteria were defined to more comprehensively capture all response patterns. Third, delayed separation of Kaplan–Meier curves in randomized immunotherapy trials can affect results. Altered statistical models describing hazard ratios as a function of time and recognizing differences before and after separation of curves may allow improved planning of phase III trials. These recommendations may improve our tools for cancer immunotherapy trials and may offer a more realistic and useful model for clinical investigation

Photodynamic therapy-generated vaccines: relevance of tumour cell death expression

Recent investigations have established that tumour cells treated in vitro by photodynamic therapy (PDT) can be used for generating potent vaccines against cancers of the same origin. In the present study, cancer vaccines were prepared by treating mouse SCCVII squamous cell carcinoma cells with photosensitiser chlorin e6-based PDT and used against poorly immunogenic SCCVII tumours growing in syngeneic immunocompetent mice. The vaccine potency increased when cells were post-incubated in culture after PDT treatment for 16 h before they were injected into tumour-bearing mice. Interfering with surface expression of phosphatidylserine (annexin V treatment) and apoptosis (caspase inhibitor treatment) demonstrated that this post-incubation effect is affiliated with the expression of changes associated with vaccine cell death. The cured mice acquired resistance to re-challenge with the same tumour, while the engagement of cytotoxic T lymphocytes was demonstrated by detection of high numbers of degranulating CD8+ cells in vaccinated tumours. The vaccines prepared from ex vivo PDT-treated SCCVII tumour tissue were also highly effective, implying that surgically removed tumour tissue can be directly used for PDT vaccines. This opens attractive prospects for employing PDT vaccines tailored for individual patients targeting specific antigens of the patient's tumour

Genetic Burden of TNNI3K in Diagnostic Testing of Patients With Dilated Cardiomyopathy and Supraventricular Arrhythmias

BACKGROUND: Genetic variants in TNNI3K (troponin-I interacting kinase) have previously been associated with dilated cardiomyopathy (DCM), cardiac conduction disease, and supraventricular tachycardias. However, the link between TNNI3K variants and these cardiac phenotypes shows a lack of consensus concerning phenotype and protein function. METHODS: We describe a systematic retrospective study of a cohort of patients undergoing genetic testing for cardiac arrhythmias and cardiomyopathy including TNNI3K. We further performed burden testing of TNNI3K in the UK Biobank. For 2 novel TNNI3K variants, we tested cosegregation. TNNI3K kinase function was estimated by TNNI3K autophosphorylation assays.RESULTS: We demonstrate enrichment of rare coding TNNI3K variants in DCM patients in the Amsterdam cohort. In the UK Biobank, we observed an association between TNNI3K missense (but not loss-of-function) variants and DCM and atrial fibrillation. Furthermore, we demonstrate genetic segregation for 2 rare variants, TNNI3K-p.Ile512Thr and TNNI3K-p.His592Tyr, with phenotypes consisting of DCM, cardiac conduction disease, and supraventricular tachycardia, together with increased autophosphorylation. In contrast, TNNI3K-p.Arg556_Asn590del, a likely benign variant, demonstrated depleted autophosphorylation. CONCLUSIONS: Our findings demonstrate an increased burden of rare coding TNNI3K variants in cardiac patients with DCM. Furthermore, we present 2 novel likely pathogenic TNNI3K variants with increased autophosphorylation, suggesting that enhanced autophosphorylation is likely to drive pathogenicity.</p