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The architecture of normal and diseased tissues strongly influences the development and progression of disease as well as responsiveness and resistance to therapy. We describe a tissue-based cyclic immunofluorescence (t-CyCIF) method for highly multiplexed immuno-fluorescence imaging of formalin-fixed, paraffin-embedded (FFPE) specimens mounted on glass slides, the most widely used specimens for histopathological diagnosis of cancer and other diseases. t-CyCIF generates up to 60-plex images using an iterative process (a cycle) in which conventional low-plex fluorescence images are repeatedly collected from the same sample and then assembled into a high-dimensional representation. t-CyCIF requires no specialized instruments or reagents and is compatible with super-resolution imaging; we demonstrate its application to quantifying signal transduction cascades, tumor antigens and immune markers in diverse tissues and tumors. The simplicity and adaptability of t-CyCIF makes it an effective method for pre-clinical and clinical research and a natural complement to single-cell genomics. To diagnose a disease such as cancer, doctors sometimes take small tissue samples called biopsies from the affected area. These biopsies are then thinly sliced and treated with dyes to identify healthy and cancerous cells. However, clinicians and scientists often need to look into what happens inside individual cells in the tissues so they can understand how cancers arise and progress. This helps them to identify different types of tumor cells and to tailor the best treatment for the patient. To do so, a number of proteins (the molecules involved in nearly all life’s processes) need to be tracked in healthy and diseased cells and tissues. This can be done thanks to a range of methods known as immunofluorescence microscopy, but following different proteins on the same slice of a sample is difficult. However, a new type of immunofluorescence known as t-CyCIF may be a solution. With this technique, a fluorescent compound is applied that will bind to a specific protein of interest. A microscope can pick up the light from the compound when the sample is imaged, which reveals the protein’s location in the cell or tissue. Then, a substance is used that deactivates the fluorescence signal. After this, another compound that binds to a new type of protein is used, and imaged. This cycle is repeated several times to locate different proteins. Lastly, the individual images are processed and stitched together to reveal the cells and their internal structures. Here, Lin, Izar et al. showed that t-CyCIF could be used to study biopsies and to obtain images that covered a large area of healthy human tissues and tumors. The technique helped to track over 60 different proteins in normal and tumor tissue samples from human patients. Several sets of experiments showed that t-CyCIF could uncover the molecular mechanisms that are disrupted during cancer, but also reveal the complexity of a single tumor. In fact, as shown with biopsies of brain cancer, cancerous cells in a tumor can be strikingly different, even when they are close to each other. Finally, the method helped to pinpoint which types of immune cells are involved in fighting a kidney tumor. Overall, such information cannot be obtained with conventional methods, yet is crucial for diagnosis and treatment. Most laboratories can readily use t-CyCIF since the technique is open source and requires equipment that is easily accessible. In fact, the technique should soon be used to assess how well certain drugs help the immune system combat cancer. Ultimately, better use of biopsies is key to customizing cancer care.

Chromosomal instability (CIN) is a driver of cancer metastasis 1 – 4 , yet the extent to which this effect depends on the immune system remains unknown. Using ContactTracing—a newly developed, validated and benchmarked tool to infer the nature and conditional dependence of cell–cell interactions from single-cell transcriptomic data—we show that CIN-induced chronic activation of the cGAS–STING pathway promotes downstream signal re-wiring in cancer cells, leading to a pro-metastatic tumour microenvironment. This re-wiring is manifested by type I interferon tachyphylaxis selectively downstream of STING and a corresponding increase in cancer cell-derived endoplasmic reticulum (ER) stress response. Reversal of CIN, depletion of cancer cell STING or inhibition of ER stress response signalling abrogates CIN-dependent effects on the tumour microenvironment and suppresses metastasis in immune competent, but not severely immune compromised, settings. Treatment with STING inhibitors reduces CIN-driven metastasis in melanoma, breast and colorectal cancers in a manner dependent on tumour cell-intrinsic STING. Finally, we show that CIN and pervasive cGAS activation in micronuclei are associated with ER stress signalling, immune suppression and metastasis in human triple-negative breast cancer, highlighting a viable strategy to identify and therapeutically intervene in tumours spurred by CIN-induced inflammation. Chromosomal instability in cancer is linked to endoplasmic reticulum stress signalling, immune suppression and metastasis, which is mediated by the cGAS–STING pathway, suppression of which can reduce metastasis.

Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks. This protocol provides guidelines for designing and validating antibody panels for fluorescence-based imaging of FFPE tissue sections using cyclic immunofluorescence (t-CyCIF) or other multiplexed imaging methods.

Treatment of BRAF ‐mutant melanomas with MAP kinase pathway inhibitors is paradigmatic of the promise of precision cancer therapy but also highlights problems with drug resistance that limit patient benefit. We use live‐cell imaging, single‐cell analysis, and molecular profiling to show that exposure of tumor cells to RAF/MEK inhibitors elicits a heterogeneous response in which some cells die, some arrest, and the remainder adapt to drug. Drug‐adapted cells up‐regulate markers of the neural crest (e.g., NGFR), a melanocyte precursor, and grow slowly. This phenotype is transiently stable, reverting to the drug‐naïve state within 9 days of drug withdrawal. Transcriptional profiling of cell lines and human tumors implicates a c‐Jun/ECM/FAK/Src cascade in de‐differentiation in about one‐third of cell lines studied; drug‐induced changes in c‐Jun and NGFR levels are also observed in xenograft and human tumors. Drugs targeting the c‐Jun/ECM/FAK/Src cascade as well as BET bromodomain inhibitors increase the maximum effect ( E max ) of RAF/MEK kinase inhibitors by promoting cell killing. Thus, analysis of reversible drug resistance at a single‐cell level identifies signaling pathways and inhibitory drugs missed by assays that focus on cell populations. Synopsis Responses of BRAF V600E melanoma cells to vemurafenib were studied at the single‐cell level using live‐cell imaging and by transcriptional and biochemical profiling to uncover a slowly dividing, de‐differentiated cell state associated with drug resistance but inhibitable by drug combinations. Cell‐to‐cell variability in BRAF V600E melanomas generates drug‐tolerant subpopulations. The drug‐tolerant, slowly dividing NFGR High state is transiently heritable. Drugs against a proposed c‐Jun/ECM/FAK/Src cascade block acquisition of this phenotype. The NGFR High drug‐tolerant state is also blocked by BET inhibitors in vitro and in vivo . Drugs that block adaptation by cell subpopulations increase cell killing by RAF/MEK inhibitors. LINCS‐compliant data and methods are freely available to enhance reproducibility. Graphical Abstract Responses of BRAF V600E melanoma cells to vemurafenib were studied at the single‐cell level using live‐cell imaging and by transcriptional and biochemical profiling to uncover a slowly dividing, de‐differentiated cell state associated with drug resistance but inhibitable by drug combinations.

We previously reported the results of a randomized phase II trial (NCT02904954) in patients with early-stage non-small cell lung cancer (NSCLC) who were treated with either two preoperative cycles of the anti-PD-L1 antibody durvalumab alone or combined with immunomodulatory doses of stereotactic radiation (DRT). The trial met its primary endpoint of major pathological response, which was significantly higher following DRT with no new safety signals. Here, we report on the prespecified secondary endpoint of disease-free survival (DFS) regardless of treatment assignment and the prespecified exploratory analysis of DFS in each arm of the trial. DFS at 2 and 3 years across patients in both arms of the trial were 73% (95% CI: 62.1–84.5) and 65% (95% CI: 52.5–76.9) respectively. For the exploratory endpoint of DFS in each arm of the trial, three-year DFS was 63% (95% CI: 46.0–80.4) in the durvalumab monotherapy arm compared to 67% (95% CI: 49.6–83.4) in the dual therapy arm. In addition, we report post hoc exploratory analysis of progression-free survival as well as molecular correlates of response and recurrence through high-plex immunophenotyping of sequentially collected peripheral blood and gene expression profiles from resected tumors in both treatment arms. Together, our results contribute to the evolving landscape of neoadjuvant treatment regimens for NSCLC and identify easily measurable potential biomarkers of response and recurrence. The authors previously reported the primary outcomes of a randomized phase II trial comparing neoadjuvant durvalumab (anti-PD-L1) alone or in combination with stereotactic radiotherapy in patients with early-stage NSCLC. Here, the authors report the secondary outcomes of the trial and post hoc analysis.

Significance The paper describes an assay for cellular transformation that involves growth in low attachment (GILA). This assay is comparable to the gold-standard soft-agar assay, but it is much easier to perform and is suitable for high-throughput drug and genetic screens. We describe such screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. Such molecules are unlikely to be found through conventional drug screening. Lastly, we demonstrate the ability of Food and Drug Administration-approved noncancer drugs to selectively kill ovarian cancer cells derived from patients with chemotherapy-resistant disease, suggesting this approach may provide useful information for personalized cancer treatment. Colony formation in soft agar is the gold-standard assay for cellular transformation in vitro, but it is unsuited for high-throughput screening. Here, we describe an assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the soft-agar assay. Using GILA, we describe high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. Such molecules are unlikely to be found through conventional drug screening, and they include kinase inhibitors and drugs for noncancer diseases. In addition to known oncogenes, the genetic screen identifies genes that contribute to cellular transformation. Lastly, we demonstrate the ability of Food and Drug Administration-approved noncancer drugs to selectively kill ovarian cancer cells derived from patients with chemotherapy-resistant disease, suggesting this approach may provide useful information for personalized cancer treatment.

Viral infection often causes severe damage to the lungs, leading to the appearance of ectopic basal cells (EBCs) and tuft cells in the lung parenchyma. Thus far, the roles of these ectopic epithelial cells in alveolar regeneration remain controversial. Here, we confirm that the ectopic tuft cells are originated from EBCs in mouse models and COVID-19 lungs. The differentiation of tuft cells from EBCs is promoted by Wnt inhibition while suppressed by Notch inhibition. Although progenitor functions have been suggested in other organs, pulmonary tuft cells don’t proliferate or give rise to other cell lineages. Consistent with previous reports, Trp63 CreERT2 and KRT5-CreERT2 -labeled ectopic EBCs do not exhibit alveolar regeneration potential. Intriguingly, when tamoxifen was administrated post-viral infection, Trp63 CreERT2 but not KRT5-CreERT2 labels islands of alveolar epithelial cells that are negative for EBC biomarkers. Furthermore, germline deletion of Trpm5 significantly increases the contribution of Trp63 CreERT2 -labeled cells to the alveolar epithelium. Although Trpm5 is known to regulate tuft cell development, complete ablation of tuft cell production fails to improve alveolar regeneration in Pou2f3 -/- mice, implying that Trpm5 promotes alveolar epithelial regeneration through a mechanism independent of tuft cells.

Spatial omics technologies can help identify spatially organized biological processes, but existing computational approaches often overlook structural dependencies in the data. Here, we introduce Smoother, a unified framework that integrates positional information into non-spatial models via modular priors and losses. In simulated and real datasets, Smoother enables accurate data imputation, cell-type deconvolution, and dimensionality reduction with remarkable efficiency. In colorectal cancer, Smoother-guided deconvolution reveals plasma cell and fibroblast subtype localizations linked to tumor microenvironment restructuring. Additionally, joint modeling of spatial and single-cell human prostate data with Smoother allows for spatial mapping of reference populations with significantly reduced ambiguity.

The lack of techniques for noninvasive imaging of inflammation has challenged precision medicine management of acute respiratory distress syndrome (ARDS). Here, we determined the potential of positron emission tomography (PET) of chemokine-like receptor-1 (CMKLR1) to monitor lung inflammation in a murine model of lipopolysaccharide- induced injury. Lung uptake of a CMKLR1-targeting radiotracer, [64Cu] NODAGA-CG34, was significantly increased in lipopolysaccharide-induced injury, correlated with the expression of multiple inflammatory markers, and reduced by dexamethasone treatment. Monocyte-derived macrophages, followed by interstitial macrophages and monocytes were the major CMKLR1-expressing leukocytes contributing to the increased tracer uptake throughout the first week of lipopolysaccharide-induced injury. The clinical relevance of CMKLR1 as a biomarker of lung inflammation in ARDS was confirmed using single-nuclei RNA-sequencing datasets which showed significant increases in CMKLR1 expression among transcriptionally distinct subsets of lung monocytes and macrophages in COVID-19 patients vs. controls. CMKLR1-targeted PET is a promising strategy to monitor the dynamics of lung inflammation and response to anti-inflammatory treatment in ARDS.

Two self-expandable stents, the Neuroform and the Enterprise stent, are widely used for stent-assisted coiling (SAC) of complex shaped intracranial aneurysms. However, comparative knowledge about technical feasibility, peri- and post-procedural morbidity and mortality, packing densities as well as follow-up data is limited. We conducted a retrospective study to investigate differences in aneurysms stented with the Enterprise or Neuroform stents. Angiographic follow-up (mean 19.42 months) was available in 72.6% (61/84) of aneurysms treated with stent-assisted coiling. We further sought to compare stent-assisted coiling to a matched patient population with aneurysms treated by conventional coil embolization. The stenting success rate of the Enterprise was higher compared to the Neuroform stent (46/48 and 42/51, respectively). In 5 of 9 cases in which the Neuroform stent was not navigable to the landing zone, we successfully deployed an Enterprise stent instead. Eventually, 42 aneurysms were coiled after stenting in each group. We observed no significant differences in peri-procedural complication rate, post-procedural hospital stay, packing density, recurrence rate or number of in-stent stenosis. Strikingly, 36.1% of followed aneurysms in the SAC group showed progressive occlusion on angiographic follow-up imaging. The packing density was significantly higher in aneurysms treated by SAC as compared to conventionally coiled aneurysms, while recanalization rate was significantly lower in the SAC group. The procedural success rate is higher using the Enterprise, but otherwise both stents exhibited similar characteristics. Lower recurrence frequency and complication rates comparable to conventional coil embolization emphasize the importance of stent-assisted coiling in the treatment of complex aneurysms. Progressive occlusion on angiographic follow-up was a distinct and frequent observation in the SAC group and may in part be due to flow diversion.