Protocol for live-cell imaging during Tumor Treating Fields treatment with Inovitro Live.

Tumor Treating Fields (TTFields) are an FDA-approved anticancer treatment using alternating electric fields. Here, we present a protocol to perform live-cell imaging (LCI) of cells during TTFields treatment with the Inovitro LiveTM system. The setup we describe dissipates TTFields-related heat production and can be used in conjunction with any LCI-compatible microscope setup. This approach will enable further elucidation of TTFields' mechanism of action at the molecular level and facilitate the development of promising combination strategies.

Breast adipocyte size associates with ipsilateral invasive breast cancer risk after ductal carcinoma in situ.

Although ductal carcinoma in situ (DCIS) is a non-obligate precursor to ipsilateral invasive breast cancer (iIBC), most DCIS lesions remain indolent. Hence, overdiagnosis and overtreatment of DCIS is a major concern. There is an urgent need for prognostic markers that can distinguish harmless from potentially hazardous DCIS. We hypothesised that features of the breast adipose tissue may be associated with risk of subsequent iIBC. We performed a case-control study nested in a population-based DCIS cohort, consisting of 2658 women diagnosed with primary DCIS between 1989 and 2005, uniformly treated with breast conserving surgery (BCS) alone. We assessed breast adipose features with digital pathology (HALO®, Indica Labs) and related these to iIBC risk in 108 women that developed subsequent iIBC (cases) and 168 women who did not (controls) by conditional logistic regression, accounting for clinicopathological and immunohistochemistry variables. Large breast adipocyte size was significantly associated with iIBC risk (odds ratio (OR) 2.75, 95% confidence interval (95% CI) = 1.25-6.05). High cyclooxygenase (COX)-2 protein expression in the DCIS cells was also associated with subsequent iIBC (OR 3.70 (95% CI = 1.59-8.64). DCIS with both high COX-2 expression and large breast adipocytes was associated with a 12-fold higher risk (OR 12.0, 95% CI = 3.10-46.3, P < 0.001) for subsequent iIBC compared with women with smaller adipocyte size and low COX-2 expression. Large breast adipocytes combined with high COX-2 expression in DCIS is associated with a high risk of subsequent iIBC. Besides COX-2, adipocyte size has the potential to improve clinical management in patients diagnosed with primary DCIS.

Labeling and tracking of immune cells in ex vivo human skin.

Human skin harbors various immune cells that are crucial for the control of injury and infection. However, the current understanding of immune cell function within viable human skin tissue is limited. We developed an ex vivo imaging approach in which fresh skin biopsies are mounted and then labeled with nanobodies or antibodies against cell surface markers on tissue-resident memory CD8+ T cells, other immune cells of interest, or extracellular tissue components. Subsequent longitudinal imaging allows one to describe the dynamic behavior of human skin-resident cells in situ. In addition, this strategy can be used to study immune cell function in murine skin. The ability to follow the spatiotemporal behavior of CD8+ T cells and other immune cells in skin, including their response to immune stimuli, provides a platform to investigate physiological immune cell behavior and immune cell behavior in skin diseases. The mounting, staining and imaging of skin samples requires ~1.5 d, and subsequent tracking analysis requires a minimum of 1 d. The optional production of fluorescently labeled nanobodies takes ~5 d.

Neoadjuvant immunotherapy leads to pathological responses in MMR-proficient and MMR-deficient early-stage colon cancers.

PD-1 plus CTLA-4 blockade is highly effective in advanced-stage, mismatch repair (MMR)-deficient (dMMR) colorectal cancers, yet not in MMR-proficient (pMMR) tumors. We postulated a higher efficacy of neoadjuvant immunotherapy in early-stage colon cancers. In the exploratory NICHE study (ClinicalTrials.gov: NCT03026140), patients with dMMR or pMMR tumors received a single dose of ipilimumab and two doses of nivolumab before surgery, the pMMR group with or without celecoxib. The primary objective was safety and feasibility; 40 patients with 21 dMMR and 20 pMMR tumors were treated, and 3 patients received nivolumab monotherapy in the safety run-in. Treatment was well tolerated and all patients underwent radical resections without delays, meeting the primary endpoint. Of the patients who received ipilimumab + nivolumab (20 dMMR and 15 pMMR tumors), 35 were evaluable for efficacy and translational endpoints. Pathological response was observed in 20/20 (100%; 95% exact confidence interval (CI): 86-100%) dMMR tumors, with 19 major pathological responses (MPRs, ≤10% residual viable tumor) and 12 pathological complete responses. In pMMR tumors, 4/15 (27%; 95% exact CI: 8-55%) showed pathological responses, with 3 MPRs and 1 partial response. CD8+PD-1+ T cell infiltration was predictive of response in pMMR tumors. These data indicate that neoadjuvant immunotherapy may have the potential to become the standard of care for a defined group of colon cancer patients when validated in larger studies with at least 3 years of disease-free survival data.

Author Correction: Tissue patrol by resident memory CD8+ T cells in human skin.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tissue patrol by resident memory CD8+ T cells in human skin.

Emerging data show that tissue-resident memory T (TRM) cells play an important protective role at murine and human barrier sites. TRM cells in the epidermis of mouse skin patrol their surroundings and rapidly respond when antigens are encountered. However, whether a similar migratory behavior is performed by human TRM cells is unclear, as technology to longitudinally follow them in situ has been lacking. To address this issue, we developed an ex vivo culture system to label and track T cells in fresh skin samples. We validated this system by comparing in vivo and ex vivo properties of murine TRM cells. Using nanobody labeling, we subsequently demonstrated in human ex vivo skin that CD8+ TRM cells migrated through the papillary dermis and the epidermis, below sessile Langerhans cells. Collectively, this work allows the dynamic study of resident immune cells in human skin and provides evidence of tissue patrol by human CD8+ TRM cells.

Expression patterns of sterol transporters NPC1 and NPC2 in the cnidarian-dinoflagellate symbiosis.

The symbiotic interaction between cnidarians (e.g., corals and sea anemones) and photosynthetic dinoflagellates of the genus Symbiodinium is triggered by both host-symbiont recognition processes and metabolic exchange between the 2 partners. The molecular communication is crucial for homeostatic regulation of the symbiosis, both under normal conditions and during stresses that further lead to symbiosis collapse. It is therefore important to identify and fully characterise the key players of this intimate interaction at the symbiotic interface. In this study, we determined the cellular and subcellular localization and expression of the sterol-trafficking Niemann-Pick type C proteins (NPC1 and NPC2) in the symbiotic sea anemones Anemonia viridis and Aiptasia sp. We first established that NPC1 is localised within vesicles in host tissues and to the symbiosome membranes in several anthozoan species. We demonstrated that the canonical NPC2-a protein is mainly expressed in the epidermis, whereas the NPC2-d protein is closely associated with symbiosome membranes. Furthermore, we showed that the expression of the NPC2-d protein is correlated with symbiont presence in healthy symbiotic specimens. As npc2-d is a cnidarian-specific duplicated gene, we hypothesised that it probably arose from a subfunctionalisation process that might result in a gain of function and symbiosis adaptation in anthozoans. Niemann-Pick type C proteins may be key players in a functional symbiosis and be useful tools to study host-symbiont interactions in the anthozoan-dinoflagellate association.

The Angiocrine Factor Rspondin3 Is a Key Determinant of Liver Zonation.

Liver zonation, the spatial separation of different metabolic pathways along the liver sinusoids, is fundamental for proper functioning of this organ, and its disruption can lead to the development of metabolic disorders such as hyperammonemia. Metabolic zonation involves the induction of ß-catenin signaling around the central veins, but how this patterned activity is established and maintained is unclear. Here, we show that the signaling molecule Rspondin3 is specifically expressed within the endothelial compartment of the central vein. Conditional deletion of Rspo3 in mice disrupts activation of central fate, demonstrating its crucial role in determining and maintaining ß-catenin-dependent zonation. Moreover, ectopic expression of Rspo1, a close family member of Rspo3, induces the expression of pericentral markers, demonstrating Rspondins to be sufficient to imprint a more central fate. Thus, Rspo3 is a key angiocrine factor that controls metabolic zonation of liver hepatocytes.

Nicotinic modulation of synaptic transmission and plasticity in cortico-limbic circuits.

Nicotine is the principle addictive agent delivered via cigarette smoking. The addictive activity of nicotine is due to potent interactions with nicotinic acetylcholine receptors (nAChRs) on neurons in the reinforcement and reward circuits of the brain. Beyond its addictive actions, nicotine is thought to have positive effects on performance in working memory and short-term attention-related tasks. The brain areas involved in such behaviors are part of an extensive cortico-limbic network that includes relays between prefrontal cortex (PFC) and cingulate cortex (CC), hippocampus, amygdala, ventral tegmental area (VTA) and the nucleus accumbens (nAcc). Nicotine activates a broad array of nAChRs subtypes that can be targeted to pre- as well as peri- and post-synaptic locations in these areas. Thereby, nicotine not only excites different types of neurons, but it also perturbs baseline neuronal communication, alters synaptic properties and modulates synaptic plasticity. In this review we focus on recent findings on nicotinic modulation of cortical circuits and their targets fields, which show that acute and transient activation of nicotinic receptors in cortico-limbic circuits triggers a series of events that affects cognitive performance in a long lasting manner. Understanding how nicotine induces long-term changes in synapses and alters plasticity in the cortico-limbic circuits is essential to determining how these areas interact in decoding fundamental aspects of cognition and reward.

Presynaptic type III neuregulin 1 is required for sustained enhancement of hippocampal transmission by nicotine and for axonal targeting of alpha7 nicotinic acetylcholine receptors.

Both the neuregulin 1 (Nrg1) and alpha7 nicotinic acetylcholine receptor (alpha7*nAChRs) genes have been linked to schizophrenia and associated sensory-motor gating deficits. The prominence of nicotine addiction in schizophrenic patients is reflected in the normalization of gating deficits by nicotine self-administration. To assess the role of presynaptic type III Nrg1 at hippocampal-accumbens synapses, an important relay in sensory-motor gating, we developed a specialized preparation of chimeric circuits in vitro. Synaptic relays from Nrg1(tm1Lwr) heterozygote ventral hippocampal slices to wild-type (WT) nucleus accumbens neurons (1) lack a sustained, alpha7*nAChRs-mediated phase of synaptic potentiation seen in comparable WT/WT circuits and (2) are deficient in targeting alpha7*nAChRs to presynaptic sites. Thus, selective alteration of the level of presynaptic type III Nrg1 dramatically affects the modulation of glutamatergic transmission at ventral hippocampal to nucleus accumbens synapses.