SUMMARY 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.

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.

39The architecture of normal and diseased tissues strongly influences the development and 40 progression of disease as well as responsiveness and resistance to therapy. We describe a tissue-based 41 cyclic immunofluorescence (t-CyCIF) method for highly multiplexed immuno-fluorescence imaging of 42 formalin-fixed, paraffin-embedded (FFPE) specimens mounted on glass slides, the most widely used 43 specimens for histopathological diagnosis of cancer and other diseases. t-CyCIF generates up to 60-plex 44 images using an iterative process (a cycle) in which conventional low-plex fluorescence images are 45 repeatedly collected from the same sample and then assembled into a high dimensional representation. t- 46 CyCIF requires no specialized instruments or reagents and is compatible with super-resolution imaging; 47 we demonstrate its application to quantifying signal transduction cascades, tumor antigens and immune 48 markers in diverse tissues and tumors. The simplicity and adaptability of t-CyCIF makes it an effective 49 method for pre-clinical and clinical research and a natural complement to single-cell genomics. 50 51 52Histopathology is among the most important and widely used methods for diagnosing human disease 53 and studying the development of multi-cellular organisms. As commonly performed, imaging of formalin-fixed, 54 paraffin-embedded (FFPE) tissue has relatively low dimensionality, primarily comprising Hematoxylin and 55 Eosin (H&E) staining supplemented by immunohistochemistry (IHC). The potential of IHC to aid in diagnosis 56 and prioritization of therapy is well established 1 but IHC is primarily a single channel method: imaging multiple 57 antigens usually involves the analysis of sequential tissue slices or harsh stripping protocols (although limited 58 multiplexing is possible using IHC and bright-field imaging 2,3 ). Antibody detection via formation of a brown 59 diamino-benzidine (DAB) or similar precipitate is also less quantitative than fluorescence 4 . The limitations of 60 IHC are particularly acute when it is necessary to quantify complex cellular states and multiple cell types, such 61 as tumor infiltrating regulatory and cytotoxic T cells, 5 in parallel with tissue and pharmaco-dynamic markers. 62 Advances in DNA and RNA profiling have dramatically improved our understanding of oncogenesis 63 and propelled the development of targeted anti-cancer drugs. 6 Sequence data are particularly useful when an 64 oncogenic driver is both a drug target and a biomarker of drug response, such as BRAF V600E in melanoma 7 or 65 BCR-ABL 8 in chronic myelogenous leukemia. However, in the case of drugs that act through cell non- 66 autonomous mechanisms, such as immune checkpoint inhibitors, tumor-drug interaction must be studied in the 67 context of multi-cellular environments that include both cancer and non-malignant stromal and infiltrating 68 immune cells. Multiple studies have established that these components of the tumor microenvironment strongly 69 influence the initiatio...