Dynamic effective elasticity of melanoma cells under shear and elongational flow confirms estimation from force spectroscopy

The detection and enrichment of circulating melanoma cells is a challenge, as the cells are very heterogeneous in terms of their biomechanical properties and surface markers. In addition, there is a lack of valid and reliable biomarkers predicting progress and therapeutic response. In this study, we analyze the elasticity of A375 melanoma cells by applying force spectroscopy and a microfluidic method. To identify and eventually separate freely circulating tumor cells, it is crucial to know their physical properties precisely. First, we use standard AFM force spectroscopy, where the elasticity of the cells is calculated from indentation with a pyramidal tip. To extend the limits of the measurements with a tip, we then use cantilevers without a tip to apply force over a larger area of the cells. The resulting Young’s moduli are slightly lower and vary less without the tip, presumably because of the spatial inhomogeneity of the cells. Finally, we implement our microfluidic method: we measure single cell elasticity by analyzing their deformation in high-speed micrographs while passing a stenosis. Combining the force field and the change in shape provides the basis for a stress–strain diagram. The results from the microfluidic deformation analysis were well in accordance with the results from force spectroscopy. The microfluidic method, however, provides advantages over conventional methods, as it is less invasive and less likely to harm the cell during the measurement. The whole cell is measured as one entity without having contact to a stiff substrate, while force spectroscopy is limited to the contact area of the tip, and in some cases dependent of the cell substrate interaction. Consequently, microfluidic deformation analysis allows us to predict the overall elastic behavior of the whole, inhomogeneous cell in three-dimensional force fields. This method may contribute to improve the detection of circulating melanoma cells in the clinical practice

The Systemic Management of Advanced Melanoma in 2016

Melanoma is a common type of skin cancer with a high propensity to metastasize. Tyrosine kinase inhibitors targeting the mitogen-activated protein kinase (MAPK) pathway and immune checkpoint blockade have recently revolutionized the management of unresectable and metastatic disease. However, acquired resistance and primary non-response to therapy require novel treatment strategies and combinations. The purpose of this review is to provide a brief and up-to-date overview on the clinical management and current trial landscape in melanoma. We summarize the most pertinent studies on BRAF/MEK inhibitors and blockade of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). Although most agents show robust antitumor efficacy as single agents, further improvements have been achieved by the combination of both approved and developing drugs. We discuss ongoing trials and evaluate future approaches that may provide additional efficacy with less toxicity. (C) 2016 S. Karger GmbH, Freibur

A Cell-Permeable Inhibitor to Trap Gαq Proteins in the Empty Pocket Conformation

In spite of the crucial role of heterotrimeric G proteins as molecular switches transmitting signals from G protein-coupled receptors, their selective manipulation with small molecule, cell-permeable inhibitors still remains an unmet challenge. Here, we report that the small molecule BIM-46187, previously classified as pan-G protein inhibitor, preferentially silences Gαq signaling in a cellular context-dependent manner. Investigations into its mode of action reveal that BIM traps Gαq in the empty pocket conformation by permitting GDP exit but interdicting GTP entry, a molecular mechanism not yet assigned to any other small molecule Gα inhibitor to date. Our data show that Gα proteins may be “frozen” pharmacologically in an intermediate conformation along their activation pathway and propose a pharmacological strategy to specifically silence Gα subclasses with cell-permeable inhibitors

Combined immune checkpoint blockade (anti-PD-1/anti-CTLA-4): Evaluation and management of adverse drug reactions

Background: Combined immune checkpoint blockade (ICB) provides unprecedented efficacy gains in numerous cancer indications, with PD-1 inhibitor nivolumab plus CTLA-4 inhibitor ipilimumab in advanced melanoma as first-ever approved therapies for combined ICB. However, gains in efficacy must be balanced against a higher frequency and severity of adverse drug reactions (ADR). Because delays in diagnosis and management might result in symptom worsening and further complications, clinicians shall be well trained to identify ADR promptly and monitor patients adequately. This paper reviews safety data assessed by the European Medicines Agency for the anti-PD-1/CTLA-4 combination and provides a literature overview on published case reports for rare ADR with suspected potential underreporting. Incidences and kinetics of immune-related ADR are described. Recommendations for the evaluation and management of ADR are convened by an interdisciplinary expert panel focusing on rare but clinically important side effects arising from combined ICB. Background: Pooled safety data from 1551 patients with advanced melanoma, treated either with 3 mg/kg ipilimumab plus 1 mg/kg nivolumab (N = 407), or nivolumab alone (N = 787), or ipilimumab alone (N = 357) demonstrate that immune-related ADR occur more frequently for the combination, with a shorter time-to-onset, and tend to be more severe. The majority of events is reversible after systemic use of glucocorticoids, notably methylprednisolone or equivalents;in certain cases of long-lasting and refractory immune toxicities, non-steroidal immunosuppressants may be used, once ICB is interrupted or terminated. Combined ICB has considerable toxicities, therefore close monitoring and high experience in diagnosis and treatment of ADR is necessary

Clinicopathologic features of primary cutaneous melanoma: a single centre analysis of a Swiss regional population

BACKGROUND: Melanoma is a common type of skin cancer with poor survival in advanced stages. Screening efforts aim to detect and tackle tumors at an early stage. However, regional population-based data at the time of initial diagnosis are sparse. OBJECTIVES: To analyse clinical and pathologic tumor characteristics in a Swiss population. MATERIALS AND METHODS: Melanoma samples diagnosed at a large Swiss academic department were evaluated for demographic, clinical and histopathologic data. RESULTS: We analysed a total of 254 melanoma samples. In situ tumors were more common in females than in males (70.6% vs. 29.4%; p = 0.0032). The acro-lentiginous subtype was more common in in situ compared to invasive tumors (14.7% vs. 5.5%; p = 0.0011). Invasive tumors showed a preference for male gender in patients beyond 60 years of age (p = 0.0080). The most frequent anatomic sites were the trunk in males and the legs in females. Regression was more common in males than in females (35.2% vs. 11.7%; p = 0.0001). Breslow's thickness correlated significantly with age but not with gender. Ulceration was common in tumors thicker than 2.01 mm (48.4%; p = 0.0001). Regression was frequently detected in melanomas thinner than 1.00 mm (29.3%; p = 0.0263). CONCLUSION: Screening efforts should target elderly patients. Skin examinations should include acral localisations and focus on the trunk in males and the lower extremities in females. Population-based analyses can help to fine-tune melanoma screening in defined regional populations

Head-to-Head Comparison of BRAF/MEK Inhibitor Combinations Proposes Superiority of Encorafenib Plus Trametinib in Melanoma

BRAFV600 mutations in melanoma are targeted with mutation-specific BRAF inhibitors in combination with MEK inhibitors, which have significantly increased overall survival, but eventually lead to resistance in most cases. Additionally, targeted therapy for patients with NRASmutant melanoma is difficult. Our own studies showed that BRAF inhibitors amplify the effects of MEK inhibitors in NRASmutant melanoma. This study aimed at identifying a BRAF and MEK inhibitor combination with superior anti-tumor activity to the three currently approved combinations. We, thus, assessed anti-proliferative and pro-apoptotic activities of all nine as well as resistance-delaying capabilities of the three approved inhibitor combinations in a head-to-head comparison in vitro. The unconventional combination encorafenib/trametinib displayed the highest activity to suppress proliferation and induce apoptosis, acting in an additive manner in BRAFmutant and in a synergistic manner in NRASmutant melanoma cells. Correlating with current clinical studies of approved inhibitor combinations, encorafenib/binimetinib prolonged the time to resistance most efficiently in BRAFmutant cells. Conversely, NRASmutant cells needed the longest time to establish resistance when treated with dabrafenib/trametinib. Together, our data indicate that the most effective combination might not be currently used in clinical settings and could lead to improved overall responses

Bystander Activation and Anti-Tumor Effects of CD8+ T Cells Following Interleukin-2 Based Immunotherapy Is Independent of CD4+ T Cell Help

<div><p>We have previously demonstrated that immunotherapy combining agonistic anti-CD40 and IL-2 (IT) results in synergistic anti-tumor effects. IT induces expansion of highly cytolytic, antigen-independent “bystander-activated” (CD8⁺CD44^high) T cells displaying a CD25⁻NKG2D⁺ phenotype in a cytokine dependent manner, which were responsible for the anti-tumor effects. While much attention has focused on CD4+ T cell help for antigen-specific CD8+ T cell expansion, little is known regarding the role of CD4+ T cells in antigen-nonspecific bystander-memory CD8+ T cell expansion. Utilizing CD4 deficient mouse models, we observed a significant expansion of bystander-memory T cells following IT which was similar to the non-CD4 depleted mice. Expanded bystander-memory CD8+ T cells upregulated PD-1 in the absence of CD4+ T cells which has been published as a hallmark of exhaustion and dysfunction in helpless CD8+ T cells. Interestingly, compared to CD8+ T cells from CD4 replete hosts, these bystander expanded cells displayed comparable (or enhanced) cytokine production, lytic ability, and in vivo anti-tumor effects suggesting no functional impairment or exhaustion and were enriched in an effector phenotype. There was no acceleration of the post-IT contraction phase of the bystander memory CD8+ response in CD4-depleted mice. The response was independent of IL-21 signaling. These results suggest that, in contrast to antigen-specific CD8+ T cell expansion, CD4+ T cell help is not necessary for expansion and activation of antigen-nonspecific bystander-memory CD8+ T cells following IT, but may play a role in regulating conversion of these cells from a central memory to effector phenotype. Additionally, the expression of PD-1 in this model appears to be a marker of effector function and not exhaustion.</p></div

CD40/IL-2 Immunotherapy induces massive expansion of bystander memory CD8+ cells and anti-tumor effects are CD8 dependent.

<p>Three C57BL/6 mice per group were treated with IT or PBS/rIgG (control) and effects on CD8+ T cell expansion were quantified by flow cytometric analysis 11 days after the initiation of therapy. For <i>in vivo</i> tumor studies one million 3LL cells were administered by s.c. injection into the flank of C57BL/6 mice seven days prior to initiation of therapy. Six to eight 3LL bearing mice were treated with IT and/or CD8+ T cell depletion to examine CD8+ dependence of anti-tumor effects. (<b>a</b>) Gating strategy for bystander memory CD8+ CD44^high NKG2D+ CD25− cells. (<b>b–e</b>) Expansion of bystander memory CD8+ T cells in the spleen and lymph nodes of IT or vehicle treated mice expressed as total numbers (<b>b,c</b>) or as a percentage of total CD8+ T cells (<b>d,e</b>). Effects of IT and/or CD8 depletion on tumor growth (<b>f</b>) and survival (<b>g</b>).</p

Anti-tumor effects of IT in CD4 knockout mice.

<p>3LL tumor bearing WT or CD4 knockout (B6.129S2-CD4^tm1Mak/J) mice were treated with IT or PBS/rIgG (control) and survival and tumor growth were measured. For <i>in vivo</i> tumor studies one million 3LL cells were administered by s.c. injection into the flank of C57BL/6 mice seven days prior to initiation of therapy. (<b>a</b>) Survival. (<b>b</b>) Mean tumor volume with SEM. (<b>c–f</b>) Growth plots of individual tumors in each group. N = 12 mice per group. (*<i>P</i><.05, **<i>P</i><.01, ***<i>P</i><.001).</p

Increased number of PD-1+ memory CD8+ T cells after IT in CD4+ T cell deficient mice.

<p>Control or CD4 deficient (depleted or knockout) C57BL/6 mice were treated with IT or PBS/rIgG (control) and PD-1 expression on memory T-cells was quantified by flow cytometric analysis 11 days after the initiation of IT. (<b>a</b>) Representative dot plots for PD-1+ gating on CD8+ CD44^high cells in the spleens of CD4+ depletion model mice. Number of PD-1+ memory (CD44^high) CD8+ T cells in spleens (<b>b,d</b>) and LNs+ (<b>c,e</b>) of IT or vehicle treated mice in CD4+ depletion (<b>b,c</b>) or CD4 knockout (<b>d,e</b>) models. Results are representative of two (CD4 knockout) or three (CD4 depletion) independent experiments with a minimum of three mice per group. (*<i>P</i><.05, **<i>P</i><.01, ***<i>P</i><.001).</p