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Suppl. F1: On-target activity and toxicity of CX-6258 Suppl. F2: On-target activity of CX-6258 and siRNA KD of Haspin Suppl. F3: Effects of CX-6258 on cell-cycle and cGAS recruitment Suppl. F4: In vivo effects of CX-6258

Suppl. Table 1: Compounds used in drug screen

Patients with melanoma resistant to RAF/MEK inhibitors (RMi) are frequently resistant to other therapies, such as immune checkpoint inhibitors (ICI), and individuals succumb to their disease. New drugs that control tumor growth and favorably modulate the immune environment are therefore needed. We report that the small-molecule CX-6258 has potent activity against both RMi-sensitive (RMS) and -resistant (RMR) melanoma cell lines. Haspin kinase (HASPIN) was identified as a target of CX-6258. HASPIN inhibition resulted in reduced proliferation, frequent formation of micronuclei, recruitment of cGAS, and activation of the cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway. In murine models, CX-6258 induced a potent cGAS-dependent type-I IFN response in tumor cells, increased IFNγ-producing CD8+ T cells, and reduced Treg frequency in vivo. HASPIN was more strongly expressed in malignant compared with healthy tissue and its inhibition by CX-6258 had minimal toxicity in ex vivo–expanded human tumor-infiltrating lymphocytes (TIL), proliferating TILs, and in vitro differentiated neurons, suggesting a potential therapeutic index for anticancer therapy. Furthermore, the activity of CX-6258 was validated in several Ewing sarcoma and multiple myeloma cell lines. Thus, HASPIN inhibition may overcome drug resistance in melanoma, modulate the immune environment, and target a vulnerability in different cancer lineages.Significance:HASPIN inhibition by CX-6258 is a novel and potent strategy for RAF/MEK inhibitor–resistant melanoma and potentially other tumor types. HASPIN inhibition has direct antitumor activity and induces a favorable immune microenvironment.

Methods and Suppl. Figure Legends

The breast is a dynamic organ whose response to physiological and pathophysiological conditions alters its disease susceptibility, yet the specific effects of these clinical variables on cell state remain poorly annotated. We present a unified, high-resolution breast atlas by integrating single-cell RNA-seq, mass cytometry, and cyclic immunofluorescence, encompassing a myriad of states. We define cell subtypes within the alveolar, hormone-sensing, and basal epithelial lineages, delineating associations of several subtypes with cancer risk factors, including age, parity, and BRCA2 germline mutation. Of particular interest is a subset of alveolar cells, termed basal-luminal (BL) cells, which exhibits poor transcriptional lineage fidelity, accumulates with age, and carries a gene signature associated with basal-like breast cancer. We further utilize a medium-depletion approach to identify molecular factors regulating cell subtype proportion in organoids. Together, these data are a rich resource to elucidate diverse mammary cell states.

Tissue-based cyclic immunofluorescence (t-CyCIF) is optimized for FFPE specimens mounted on glass slides. Dewaxing and antigen retrieval are important steps to remove wax and expose antigenic sites. This protocol describes dewaxing and antigen retrieval on a Leica Bond RX automated slide processor; similar instruments are manufactured by Ventana or Dako and are commonly found in histopathology core facilities. t-CyCIF can also be performed following manual de-waxing and antigen retrieval (e.g. microwaving slides in citrate buffer or using a pressure cooker).

Malignant abdominal fluid (ascites) frequently develops in women with advanced high-grade serous ovarian cancer (HGSOC) and is associated with drug resistance and a poor prognosis1. To comprehensively characterize the HGSOC ascites ecosystem, we used single-cell RNA sequencing to profile ~11,000 cells from 22 ascites specimens from 11 patients with HGSOC. We found significant inter-patient variability in the composition and functional programs of ascites cells, including immunomodulatory fibroblast sub-populations and dichotomous macrophage populations. We found that the previously described immunoreactive and mesenchymal subtypes of HGSOC, which have prognostic implications, reflect the abundance of immune infiltrates and fibroblasts rather than distinct subsets of malignant cells2. Malignant cell variability was partly explained by heterogeneous copy number alteration patterns or expression of a stemness program. Malignant cells shared expression of inflammatory programs that were largely recapitulated in single-cell RNA sequencing of ~35,000 cells from additionally collected samples, including three ascites, two primary HGSOC tumors and three patient ascites-derived xenograft models. Inhibition of the JAK/STAT pathway, which was expressed in both malignant cells and cancer-associated fibroblasts, had potent anti-tumor activity in primary short-term cultures and patient-derived xenograft models. Our work contributes to resolving the HSGOC landscape3-5 and provides a resource for the development of novel therapeutic approaches.

Patients with melanoma resistant to RAF/MEK inhibitors (RMi) are frequently resistant to other therapies, such as immune checkpoint inhibitors (ICI), and individuals succumb to their disease. New drugs that control tumor growth and favorably modulate the immune environment are therefore needed. We report that the small-molecule CX-6258 has potent activity against both RMi-sensitive (RMS) and -resistant (RMR) melanoma cell lines. Haspin kinase (HASPIN) was identified as a target of CX-6258. HASPIN inhibition resulted in reduced proliferation, frequent formation of micronuclei, recruitment of cGAS, and activation of the cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway. In murine models, CX-6258 induced a potent cGAS-dependent type-I IFN response in tumor cells, increased IFNγ-producing CD8+ T cells, and reduced Treg frequency in vivo. HASPIN was more strongly expressed in malignant compared with healthy tissue and its inhibition by CX-6258 had minimal toxicity in ex vivo–expanded human tumor-infiltrating lymphocytes (TIL), proliferating TILs, and in vitro differentiated neurons, suggesting a potential therapeutic index for anticancer therapy. Furthermore, the activity of CX-6258 was validated in several Ewing sarcoma and multiple myeloma cell lines. Thus, HASPIN inhibition may overcome drug resistance in melanoma, modulate the immune environment, and target a vulnerability in different cancer lineages. Significance: HASPIN inhibition by CX-6258 is a novel and potent strategy for RAF/MEK inhibitor–resistant melanoma and potentially other tumor types. HASPIN inhibition has direct antitumor activity and induces a favorable immune microenvironment.

Synovial sarcoma (SyS) is an aggressive neoplasm driven by the SS18–SSX fusion, and is characterized by low T cell infiltration. Here, we studied the cancer–immune interplay in SyS using an integrative approach that combines single-cell RNA sequencing (scRNA-seq), spatial profiling and genetic and pharmacological perturbations. scRNA-seq of 16,872 cells from 12 human SyS tumors uncovered a malignant subpopulation that marks immune-deprived niches in situ and is predictive of poor clinical outcomes in two independent cohorts. Functional analyses revealed that this malignant cell state is controlled by the SS18–SSX fusion, is repressed by cytokines secreted by macrophages and T cells, and can be synergistically targeted with a combination of HDAC and CDK4/CDK6 inhibitors. This drug combination enhanced malignant-cell immunogenicity in SyS models, leading to induced T cell reactivity and T cell–mediated killing. Our study provides a blueprint for investigating heterogeneity in fusion-driven malignancies and demonstrates an interplay between immune evasion and oncogenic processes that can be co-targeted in SyS and potentially in other malignancies. Single-cell transcriptional profiling of primary human synovial sarcoma tumors suggests that combinatorial treatment with HDAC and CDK4/CDK6 inhibitors could enhance tumor immunogenicity.

From the perspective of improving the self-healing method in construction, a tubular healing fiber was adopted as a container to improve the encapsulation capacity, which was available using a micro-capsule as a container. Knowing the direction of the stresses to which structure members are subjected, this research investigated the influence of aligning tubular healing fibers parallel to intended stress into a cementitious composite to increase the self-healing capability. For that, a healing agent was encapsulated into a tubular healing fiber made with polyvinylidene of fluoride resin (PVDF). Then, the healing fiber was combined with steel fibers to align both fibers together parallel to the direction of an intended splitting tensile stress when subjected to a magnetic field in a cylindrical cementitious composite. The alignment method and the key point through which the alignment of the healing fibers could efficiently improve autonomic self-healing were investigated. Since the magnetic field is known to be able to drag steel to an expected direction, steel fibers were combined with the healing fibers to form a hybrid fiber that aligned both fibers together. The required mixture workability was investigated to avoid the sinking of the healing fibers into the mixture. The healing efficiency, according to the orientation of the healing fibers in the composite matrix, was evaluated through a permeability test and a repetitive splitting tensile test. The aligned healing fibers performed better than the randomly distributed healing fibers. However, according to the healing efficiency with aligned healing fibers, it was deduced that the observed decreasing effect of the container’s alignment on the specimen’s mechanical properties was low enough to be neglected.

Synovial sarcoma is an aggressive mesenchymal neoplasm, driven by the SS18-SSX fusion, and characterized by immunogenic antigens expression and exceptionally low T cell infiltration levels. To study the cancer-immune interplay in this disease, we profiled 16,872 cells from 12 human synovial sarcoma tumors using single-cell RNA-sequencing (scRNA-Seq). Synovial sarcoma manifests antitumor immunity, high cellular plasticity and a core oncogenic program, which is predictive of low immune levels and poor clinical outcomes. Using genetic and pharmacological perturbations, we demonstrate that the program is controlled by the SS18-SSX driver and repressed by cytokines secreted by macrophages and T cells in the tumor microenvironment. Network modeling predicted that SS18-SSX promotes the program through HDAC1 and CDK6. Indeed, the combination of HDAC and CDK4/6 inhibitors represses the program, induces immunogenic cell states, and selectively targets synovial sarcoma cells. Our study demonstrates that immune evasion, cellular plasticity, and cell cycle are co-regulated and can be co-targeted in synovial sarcoma and potentially in other malignancies.

Tissue-based cyclic immunofluorescence (t-CyCIF) is optimized for FFPE specimens mounted on glass slides. Dewaxing and antigen retrieval are important steps to remove wax and expose antigenic sites. This protocol describes dewaxing and antigen retrieval on a Leica Bond RX automated slide processor; similar instruments are manufactured by Ventana or Dako and are commonly found in histopathology core facilities. t-CyCIF can also be performed following manual de-waxing and antigen retrieval (e.g. microwaving slides in citrate buffer or using a pressure cooker).

Balancing the potential for durable remissions with autoimmune-like toxicities is a key clinical challenge in the use of immune checkpoint inhibitors (ICI). Certain toxicities are associated with an increased response rate; however, the molecular underpinnings of this association are poorly understood. Here, we report a patient with wide spread uveal melanoma who had an exceptional response to treatment with ipilimumab and nivolumab, but suffered severe immune-related sequelae, including central serous retinopathy with retinal detachment, tinnitus, and vitiligo resembling Vogt-Koyanagi-Harada disease, and refractory enteritis. TCR-sequencing of the primary tumor, a hepatic metastasis, duodenal biopsy and peripheral blood mononuclear cells, identified the identical T cell clone in all four tissues. This case provides preliminary evidence for cross-reactivity as a mechanism for the association between effect and toxicity of ICIs. Electronic supplementary material The online version of this article (10.1186/s40425-019-0533-0) contains supplementary material, which is available to authorized users.

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 immunofluorescence imaging of specimens mounted on glass slides. 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. Key resources, reagents, and software are listed at the HMS LINCS Center Publication Page http://lincs.hms.harvard.edu/lin-elife-2018/ (RRID:SCR_016370).This page provides links to an OMERO image database from which individual images can be obtained; stitched and registered image panels can be obtained at www.cycif.org (RRID:SCR_016267) and a video illustrating the t-CyCIF method can be found at https://vimeo.com/269885646. This protocol is used in the following manuscripts: Lin J-R, Izar B, Wang S, Yapp C, Mei S, Shah P, Santagata S, Sorger PK. (2018). Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes. eLife. PMID: 29993362 Du Z, Lin JR, Rashid R, Maliga Z, Wang S, Aster J, Izar B, Sorger PK, Santagata S. (2019). Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging. Nature Protocols. PMID: 31534232 The original t-CyCIF protocol can be found at dx.doi.org/10.17504/protocols.io.rpxd5pn.

Supplemental Appendix. (DOCX 7 kb)

Background Immunohistochemical (IHC) evaluation has shown that human epidermal growth factor receptor 2 (HER2) may not be expressed homogeneously among all cancer cells within a given tumor. The clinical significance of intratumoral HER2 heterogeneity is unclear. Exploration of tumor heterogeneity is facilitated by tissue imaging technologies such as t-CyCIF, a highly multiplexed immunofluorescence microscopy technique that permits visualization of up to 60 antigens and analysis on a single cell level from formalin-fixed, paraffin-embedded tissue. To utilize t-CyCIF for the evaluation of breast tumors, this study was undertaken to: 1) validate antibodies to be used against the clinically relevant markers HER2, estrogen receptor (ER) and progesterone receptor (PR), and 2) use these antibodies along with other validated antibodies to define the tumor microenvironment (TME) to interrogate breast tumors at a single cell level. Methods T-CyCIF is an iterative whole-slide imaging process, in which successive four-channel images, each involving different antibodies, are collected from the same sample and then merged to generate a high-dimensional representation used for visualization and analysis. In phase one of this study, 948 tissue cores (representing 295 patients in triplicate) were used to validate HER2, ER, and PR antibodies against a single antibody commonly used in clinical practice as a reference. Analyses were performed at the level of tissue cores, cells and pixels. Inter-assay analyses were performed comparing: t-CyCIF vs. IHC, the latter assessed by digital pathology and two pathologists; and also, t-CyCIF vs. fluorescence in situ hybridization (FISH) for HER2. In the second phase, following selection of validated HER2, ER and PR antibodies, expression of CD45, CD68, PD-L1, p53, Ki67, pRB and the androgen receptor (AR) were evaluated at a single cell level in 312 HER2+ invasive breast cancer samples, representing 104 patients, to better understand the TME, cancer cell heterogeneity and the cell identities/states present in breast carcinomas. Results In the first phase of the study, 13 different ER, PR or HER2 antibodies were analyzed. The pixel-to-pixel evaluation, which evaluates concordance in staining, resulted in r scores of 0.86 (ER; Pearson correlation), 0.93 (PR) and 0.94 (HER2) and correlation scores in single-cell comparisons ranged from 0.76 to 0.81. Correlation scores on the tissue core level were high in the inter-assay analyses, i.e. t-CyCIF vs. IHC (e.g. r scores up to 0.87 and 0.91 for ER and HER2, respectively, on t-CyCIF vs. Aperio; and 0.85 to 0.94 by pathology review) and t-CyCIF vs. HER2 FISH (r scores up to 0.71). This resulted in validated fluorophore-conjugated antibody panels for use in t-CyCIF that correspond well to established standards. In the second phase, single cell analysis of HER2+ breast cancer was performed. Cancer cells were defined as keratin positive and using t-Distributed Stochastic Neighbor Embedding (t-SNE) seven cancer cell clusters were identified including two HER2hi clusters differing in ER, p53, AR and PD-L1 expression, two HER2lo clusters differing in PR, Ki67, pRB, p53 and AR and three HER2neg clusters differing in PR, Ki67, ER, PD-L1 and AR. Heterogeneity scores were calculated based on diversity among clusters. Conclusion This study is the first to evaluate the performance of breast cancer-specific antibodies in a highly multiplexed imaging platform such as t-CyCIF. Using the validated antibody panel, we uncovered patterns of expression of markers relevant to breast cancer biology that correlate with HER2 high, low and negative states. Ongoing studies are looking at correlations between HER2 heterogeneity, responses to therapy and clinical outcomes. Citation Format: Jennifer L Guerriero, Jia-Ren Lin, Ricardo G Pastorello, Ziming Du, Shaolin Mei, Krishan Taneja, Stuart J Schnitt, Deborah A Dillon, Peter K Sorger, Sandro Santagata, Elizabeth A Mittendorf. Highly multiplexed tissue-based cyclic immunofluorescence (t-CyCIF) for precision oncology identifies novel patterns of HER2 heterogeneity in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-02.

Immune checkpoint inhibitors (ICI) produce durable responses in some melanoma patients, but many patients derive no clinical benefit, and the molecular underpinnings of such resistance remain elusive. Here, we leveraged single-cell RNA-seq (scRNA-seq) from 33 melanoma tumors and computational analyses to interrogate malignant cell states that promote immune evasion. We identified a resistance program expressed by malignant cells that is associated with T-cell exclusion and immune evasion. The program is expressed prior to immunotherapy, characterizes cold niches in situ, and predicts clinical responses to anti-PD-1 therapy in an independent cohort of 112 melanoma patients. CDK4/6-inhibition represses this program in individual malignant cells, induces senescence, and reduces melanoma tumor outgrowth in mouse models in vivo when given in combination with immunotherapy. Our study provides a high-resolution landscape of ICI resistant cell states, identifies clinically predictive signatures, and suggests new therapeutic strategies to overcome immunotherapy resistance. This study will be published on Nov. 1st in Cell (Jerby-Arnon et al., Cell 2018). This abstract is also being presented as Poster A25. Citation Format: Parin Shah, Michael Cuoco, Mei-Ju Su, Johannes Melms, Rachel Leeson, Abhay Kanodia, Shaolin Mei, Jia-Ren Lin, Shu Wang, Bokang Rabasha, David Liu, Alex K. Shalek, Itay Tirosh, Peter K. Sorger, Kai Wucherpfennig, Eliezer M. Van Allen, Dirk Schadendorf, Bruce E. Johnson, Asaf Rotem, Orit Rozenblatt-Rosen, Levi A. Garraway, Charles H. Yoon, Benjamin Izar, Aviv Regev, Livnat Jerby-Arnon. A cancer cell program promotes T-cell exclusion and resistance to checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR01.

Background: Tissue-based cyclic immunofluorescence (t-CyCIF) is a recently described technique for highly multiplexed immunofluorescence microscopy of formalin-fixed, paraffin-embedded (FFPE) specimens. Via an iterative process, successive four-channel images are collected from the same sample and then registered to each other to generate a high-dimensional representation that is used for visualization and analysis. This technique can be used to capture up to 60 different antigens on a single FFPE tumor section and permits quantification of cell lineage and state markers, intracellular signaling proteins, drug targets and immune cell antigens, thereby promoting biomarker discovery efforts that are fundamental to precision oncology. As with most technologies that utilize immunostaining, proper antibody validation is key to reliable performance. In this study we used t-CyCIF to evaluate multiple antibodies directed against proteins commonly used to characterize breast carcinomas and their associated microenvironment. Our goal was to validate these antibodies on the t-CyCIF platform prior to the more widespread use of this technology to propel novel discoveries on breast cancer initiation, progression and treatment. Methods: To choose the optimal antibody candidate for each biomarker in t-CyCIF, we compared multiple fluorophore-conjugated antibodies for each of the following three proteins routinely evaluated in breast carcinomas: estrogen receptor (ER), progesterone receptor (PR) and HER2. For each of these, a single antibody commonly used in clinical practice was used as a reference. Analyses were performed at the level of pixels, cells and tissue cores. In addition, inter-assay analyses were performed comparing: (1) t-CyCIF vs. immunohistochemistry (IHC), the latter assessed both by digital pathology and by two independent pathologists; and (2) t-CyCIF vs. fluorescence in situ hybridization (FISH) for HER2. Following validation of these antibodies, we evaluated the expression of CD45, CD68, PD-L1, p53, Ki67 and androgen receptor along with ER, PR and HER2, to better understand both the tumor microenvironment and the cell identities/states in breast carcinomas. Results: A total of 948 tissue cores were included in the study. In the first phase, 13 different antibodies were analyzed: three raised against ER and five each against PR and HER2. The pixel-to-pixel evaluation resulted in r scores using Pearson correlation equal to 0.86 for both ER markers tested; ranging from 0.88 to 0.93 for PR; and from 0.56 to 0.94 for HER2. The correlation scores in single-cell comparisons ranged from 0.76 to 0.88 for ER, 0.54 to 0.81 for PR, and from 0.56 to 0.76 for HER2. Comparisons were then performed at the tissue core level. In light of the data generated through these multiple levels of analyses, we identified fluorophore-conjugated candidates for use in t-CyCIF. Correlation scores on the tissue core level were high in the inter-assay analyses, i.e. t-CyCIF vs. IHC (e.g. r scores up to 0.91 for HER2 on t-CyCIF vs. IHC) and t-CyCIF vs. HER2 FISH (r scores up to 0.71). In the second phase, we characterized the tumor microenvironment and cell identities present in 260 breast carcinomas. With a qualified panel of antibodies, we performed single-cell analyses of 589,343 cells and identified unexpected patterns of PD-L1 expression in distinct populations of tumor cells. Conclusion: This study is the first to evaluate the performance of breast cancer-specific antibodies in a highly multiplexed imaging platform such as t-CyCIF. This work demonstrates a step-by-step approach for qualifying reagents to be used in a multiplexed, spatially resolved tissue imaging modality. This validation study will facilitate the use of t-CyCIF for additional studies in breast cancer to evaluate both tumor elements and components of the microenvironment. Citation Format: Ricardo G Pastorello, Jia-Ren Lin, Ziming Du, Shaolin Mei, Krishan Taneja, Deborah A Dillon, Stuart J Schnitt, Peter K Sorger, Elizabeth A Mittendorf, Sandro Santagata, Jennifer L Guerriero. Highly multiplexed tissue-based cyclic immunofluorescence in breast cancer for precision oncology [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-02-03.

Ubiquitination and its reverse process, deubiquitination, play essential roles in neural development, function, and plasticity. A20, a ubiquitin editing enzyme that can remove K63-polyubiquitin chains from substrates and attach K48-polyubiquitin chains to them, is a critical component in the NF-κB signaling pathway in the immune system. This dual ubiquitin enzyme is also present in mammalian brains, but its potential role in neurons and synapses is unknown. We show that A20 in pyramidal neurons potently regulates dendritic arborization, spine morphogenesis, and synaptic transmission through an NF-κB-dependent mechanism. In cultured hippocampal neurons, overexpression of A20 reduced dendritic complexity and spine size and density, whereas A20 knockdown increased spine size and density, as well as clustering of the postsynaptic scaffold PSD-95 and glutamate receptor subunit GluA1. A20 effects in vitro were recapitulated in vivo where increasing or decreasing A20 expression in mouse brains reduced and enhanced spine density, respectively. Functionally, A20 knockdown significantly increased the amplitude, but not frequency of miniature excitatory postsynaptic currents, suggesting a role in postsynaptic efficacy. A20 negatively regulated NF-κB activation in neurons and A20 mutants deficient in either the deubiquitinase or the ubiquitin ligase activity failed to suppress NF-κB activation or reduce spine morphogenesis. Finally, selective inhibition of NF-κB abolished A20 knockdown-elicited spine formation, suggesting that A20 exerts its modulation on synapses through NF-κB signaling. Together, our study reveals a previously unknown role for A20, the only known ubiquitin editing enzyme with both deubiquitinase and ubiquitin ligase activity, in dendritic arborization, spine remodeling, and synaptic plasticity.

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.

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., eLife digest 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.

Immune checkpoint inhibitors (ICI) produce durable responses in some melanoma patients, but many patients derive no clinical benefit. The molecular underpinnings of ICI resistance involve intricate cell-cell interactions that are yet elusive. To systematically map the interactions between malignant and immune cells in the tumor ecosystem, we applied single-cell RNA sequencing to 31 human melanoma tumors, profiling thousands of malignant, immune, and stromal cells. We identified a transcriptional program in malignanT-cells that is strongly associated with T-cell exclusion and immunotherapy resistance. Using highly multiplexed in situ imaging we first demonstrated that this program characterizes malignanT-cells in “cold” niches. Next, we showed that the program predicts clinical responses to ICI according to multiple independent validation cohorts, including a new cohort that we obtained from 112 melanoma patients treated with anti-PD-1 therapy. We then identified CDK4/6 as master regulators of this resistance program, and found that CDK4/6 inhibitors repress the program and shift melanoma cells into a senescence-associated secretory phenotype. Lastly, we showed that CDK4/6-inhibition leads to a substantial reduction in melanoma tumor outgrowth in a B16 mouse model when given in combination with immunotherapy. Taken together, our study provides a high-resolution landscape of ICI-resistant cell states, identifies clinically predictive signatures, and forms a basis for the development of novel therapeutic strategies that could overcome immunotherapy resistance. Citation Format: Livnat Jerby, Parin Shah, Michael S. Cuoco, Christopher Rodman, Mei-Ju Su, Johannes M. Melms, Rachel Leeson, Abhay Kanodia, Shaolin Mei, Jia-Ren Lin, Shu Wang, Bokang Rabasha, David Liu, Gao Zhang, Claire Margolais, Orr Ashenberg, Patrick A. Ott, Elizabeth I. Buchbinder, Riz Haq, Stephen Hodi, Genevieve M. Boland, Ryan J. Sullivan, Dennie Frederick, Benchun Miao, Tabea Moll, Keith Flaherty, Meenhard Herlyn, Russell S. Jenkins, Rohit Thummalapalli, Monika S. Kowalczyk, Israel Canadas, Bastian Schilling, Adam N.R Cartwright, Adrienne M. Luoma, Shruti Malu, Patrick Hwu, Chantale Bernatchez, Marie-Andree Forget, David A. Barbie, Alex K. Shalek, Itay Tirosh, Peter K. Sorger, Kai W. Wucherpfennig, Eliezer M. Van Allen, Dirk Schadendorf, Bruce E. Johnson, Asaf Rotem, Orit Rosenblatt-Rozen, Levi A. Garraway, Charles H. Yoon, Benjamin Izar, Aviv Regev. Single-cell RNA-sequencing of metastatic melanoma identifies a cancer cell-intrinsic program associated with immune checkpoint inhibitor resistance [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A082.

Immune checkpoint inhibitors (ICIs) produce durable responses in some melanoma patients, but many patients derive no clinical benefit, and the molecular underpinnings of such resistance remain elusive. Here, we leveraged single-cell RNA sequencing (scRNA-seq) from 33 melanoma tumors and computational analyses to interrogate malignant cell states that promote immune evasion. We identified a resistance program expressed by malignant cells that is associated with T cell exclusion and immune evasion. The program is expressed prior to immunotherapy, characterizes cold niches in situ, and predicts clinical responses to anti-PD-1 therapy in an independent cohort of 112 melanoma patients. CDK4/6-inhibition represses this program in individual malignant cells, induces senescence, and reduces melanoma tumor outgrowth in mouse models in vivo when given in combination with immunotherapy. Our study provides a high-resolution landscape of ICI-resistant cell states, identifies clinically predictive signatures, and suggests new therapeutic strategies to overcome immunotherapy resistance. Single-cell sequencing of checkpoint-inhibitor-resistant melanomas identifies predictive signatures to guide therapeutic approaches to overcome immunotherapy resistance., National Cancer Institute (U.S.) (Grant K08-CA222663)

Intermediate neural progenitor cells (INPs) need to avoid differentiation and cell cycle exit while maintaining restricted developmental potential, but mechanisms preventing differentiation and cell cycle exit of INPs are not well understood. In this study, we report that the Drosophila homolog of mammalian Sp8 transcription factor Buttonhead (Btd) prevents premature differentiation and cell cycle exit of INPs in Drosophila larval type II neuroblast (NB) lineages. We show that the loss of Btd leads to elimination of mature INPs due to premature differentiation of INPs into terminally dividing ganglion mother cells. We provide evidence to demonstrate that Btd prevents the premature differentiation by suppressing the expression of the homeodomain protein Prospero in immature INPs. We further show that Btd functions cooperatively with the Ets transcription factor Pointed P1 to promote the generation of INPs. Thus, our work reveals a critical mechanism that prevents premature differentiation and cell cycle exit of Drosophila INPs. DOI: http://dx.doi.org/10.7554/eLife.03596.001, eLife digest Whereas the majority of cells in the brain are unable to divide to produce new cells, neural stem cells can divide numerous times and have the potential to become many different types of brain cells. However, in between these two extremes there is another group of cells called neural progenitors. These cells can give rise to multiple types of neurons but, in contrast to stem cells, they can undergo only a limited number of divisions. Many of the molecular mechanisms by which stem cells give rise to progenitors are similar in mammals and in the fruit fly Drosophila. In the brains of fruit fly larvae, neural stem cells called neuroblasts give rise to ‘intermediate neural progenitors', each of which can divide between four and six times. Every division generates a replacement intermediate progenitor and a cell called a GMC, which divides one last time to produce two brain cells. Intermediate progenitors must be tightly regulated to ensure that they undergo an appropriate number of divisions: too few divisions will result in a shortage of cells, disrupting brain development, whereas too many divisions will result in the formation of tumors. Now, using Drosophila brains in the laboratory, Xie et al.—and, independently, Komori et al.—have shown that a protein called ‘Buttonhead’ is responsible for maintaining this balance. Xie et al. show that deletion of the gene for Buttonhead gene caused the progenitor cells to become GMCs before they had undergone the correct number of divisions. Further experiments revealed that Buttonhead prevents this problem by suppressing a protein called Prospero. The mammalian equivalent of Buttonhead—a protein called Sp8—can substitute for Buttonhead in Drosophila neural progenitors, suggesting that the observed mechanisms may also apply to mammals. Further work is required to test this possibility directly and to examine the involvement of Sp8 in brain development and tumor formation. DOI: http://dx.doi.org/10.7554/eLife.03596.002