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Advanced solid cancers are complex assemblies of tumor, immune, and stromal cells characterized by high intratumoral variation. We use highly multiplexed tissue imaging, 3D reconstruction, spatial statistics, and machine learning to identify cell types and states underlying morphological features of known diagnostic and prognostic significance in colorectal cancer. Quantitation of these features in high-plex marker space reveals recurrent transitions from one tumor morphology to the next, some of which are coincident with long-range gradients in the expression of oncogenes and epigenetic regulators. At the tumor invasive margin, where tumor, normal, and immune cells compete, T cell suppression involves multiple cell types and 3D imaging shows that seemingly localized 2D features such as tertiary lymphoid structures are commonly interconnected and have graded molecular properties. Thus, while cancer genetics emphasizes the importance of discrete changes in tumor state, whole-specimen imaging reveals large-scale morphological and molecular gradients analogous to those in developing tissues. [Display omitted] • Multiplexed analysis shows intermixed tumor morphologies and molecular gradients • Various cancer characteristic cellular features are large, interconnected structures • 3D TLS networks show intra-TLS patterning variation • PD1-PDL1 interactions are primarily between T and myeloid cells in this CRC cohort Multiplexed whole-slide imaging analysis characterizes intermixed and graded morphological and molecular features in human colorectal cancer samples, highlighting large-scale cancer characteristic structural features and variations in intra-tertiary lymphoid cellular compositions and structural patterning. [ABSTRACT FROM AUTHOR]

Ovarian cancer is resistant to immunotherapy, and this is influenced by the immunosuppressed tumor microenvironment (TME) dominated by macrophages. Resistance is also affected by intratumoral heterogeneity, whose development is poorly understood. To identify regulators of ovarian cancer immunity, we employed a spatial functional genomics screen (Perturb-map), focused on receptor/ligands hypothesized to be involved in tumor-macrophage communication. Perturb-map recapitulated tumor heterogeneity and revealed that interleukin-4 (IL-4) promotes resistance to anti-PD-1. We find ovarian cancer cells are the key source of IL-4, which directs the formation of an immunosuppressive TME via macrophage control. IL-4 loss was not compensated by nearby IL-4-expressing clones, revealing short-range regulation of TME composition dictating tumor evolution. Our studies show heterogeneous TMEs can emerge from localized altered expression of cancer-derived cytokines/chemokines that establish immune-rich and immune-excluded neighborhoods, which drive clone selection and immunotherapy resistance. They also demonstrate the potential of targeting IL-4 signaling to enhance ovarian cancer response to immunotherapy. [Display omitted] • Perturb-map enables CRISPR screens modeling inter- and intratumor heterogeneity • Regional differences in cytokine expression create immunosuppressed tumor neighborhoods • Cancer-derived CCL7 acts locally to organize immune composition around tumor clones • Cancer cell-produced IL-4 mediates resistance to immunotherapy via macrophage control Cancer-derived paracrine factors create distinct "neighborhoods" in the tumor microenvironment, promoting clonal selection and tumor heterogeneity. In ovarian cancer, IL-4 originating from a minority of cells within the tumor can promote immune suppression and tumor resistance to immune checkpoint blockade therapy. [ABSTRACT FROM AUTHOR]

Hidradenitis suppurativa (HS) is a chronic, debilitating inflammatory skin disease characterized by keratinized epithelial tunnels that grow deeply into the dermis. Here, we examined the immune microenvironment within human HS lesions. Multi-omics profiling and multiplexed imaging identified tertiary lymphoid structures (TLSs) near HS tunnels. These TLSs were enriched with proliferative T cells, including follicular helper (Tfh), regulatory (Treg), and pathogenic T cells (IL17A + and IFNG +), alongside extensive clonal expansion of plasma cells producing antibodies reactive to keratinocytes. HS fibroblasts express CXCL13 or CCL19 in response to immune cytokines. Using a microfluidic system to mimic TLS on a chip, we found that HS fibroblasts critically orchestrated lymphocyte aggregation via tumor necrosis factor alpha (TNF-α)-CXCL13 and TNF-α-CCL19 feedback loops with B and T cells, respectively; early TNF-α blockade suppressed aggregate initiation. Our findings provide insights into TLS formation in the skin, suggest therapeutic avenues for HS, and reveal mechanisms that may apply to other autoimmune settings, including Crohn's disease. [Display omitted] • TLSs near tunnel epithelium of HS lesional skin support local immune cell proliferation • Extensive clonal expansion of plasma cells producing antibodies against keratinocytes • HS fibroblasts promote lymphocyte aggregation through TNF-α-CXCL13 and TNF-α-CCL19 loops • TNF-α blockade is more effective at the initiation of lymphocyte aggregation Hidradenitis suppurativa (HS) is a severe chronic inflammatory disease without effective treatment options. Yu et al. identify and characterize tertiary lymphoid structures (TLSs) in HS, highlighting fibroblast-driven TLS formation via cytokine feedback loops with immune cells and lymphocyte clonal expansion and antibody production against keratinocytes. Their findings have implications for autoimmune pathogenesis and treatment options. [ABSTRACT FROM AUTHOR]

Lymphocytes are key for immune surveillance of tumors, but our understanding of the spatial organization and physical interactions that facilitate lymphocyte anti-cancer functions is limited. We used multiplexed imaging, quantitative spatial analysis, and machine learning to create high-definition maps of lung tumors from a Kras / Trp53 -mutant mouse model and human resections. Networks of interacting lymphocytes ("lymphonets") emerged as a distinctive feature of the anti-cancer immune response. Lymphonets nucleated from small T cell clusters and incorporated B cells with increasing size. CXCR3-mediated trafficking modulated lymphonet size and number, but T cell antigen expression directed intratumoral localization. Lymphonets preferentially harbored TCF1+ PD-1+ progenitor CD8+ T cells involved in responses to immune checkpoint blockade (ICB) therapy. Upon treatment of mice with ICB or an antigen-targeted vaccine, lymphonets retained progenitor and gained cytotoxic CD8+ T cell populations, likely via progenitor differentiation. These data show that lymphonets create a spatial environment supportive of CD8+ T cell anti-tumor responses. [Display omitted] • Interacting networks of lymphocytes (lymphonets) from in the KP GEMM of lung cancer • Small lymphonets have mostly T cells, and B cell fraction rises as networks enlarge • A key feature of lymphonets is that they contain TCF1+PD-1+CD8+ T cell progenitors • Lymphonets gain cytotoxic CD8+ T cells after immunotherapy Gaglia et al. find striking changes in the spatial arrangement of immune cells in response to tumor antigens. T and B cells are recruited in lymphocyte networks ("lymphonets"), which contain progenitor T cells. After immunotherapy, lymphonets gain cytotoxic T cells, likely due to progenitor cell differentiation and activation in this distinct immune environment. [ABSTRACT FROM AUTHOR]

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 exhibit poor transcriptional lineage fidelity, accumulate with age, and carry 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. [Display omitted] • Multimodal single-cell analyses identify breast epithelial and stromal subtypes • Spatially distinct epithelial subsets are linked with age, parity, and BRCA2 status • Alveolar cells with poor transcriptional lineage fidelity accumulate with age • Subtypes of the three major epithelial lineages are maintained in organoid cultures Gray, Li, Rosenbluth, Selfors et al. generate a multi-dimensional atlas of breast tissues and organoids. Distinct epithelial subtypes are found to be associated with age, parity, and BRCA2 mutation. An alveolar subset termed basal-luminal cells displays poor transcriptional lineage fidelity and gene expression signatures associated with aggressive basal-like breast cancers. [ABSTRACT FROM AUTHOR]

High-Grade Serous Ovarian Cancer (HGSOC) originates from fallopian tube (FT) precursors. However, the molecular changes that occur as precancerous lesions progress to HGSOC are not well understood. To address this, we integrated high-plex imaging and spatial transcriptomics to analyze human tissue samples at different stages of HGSOC development, including p53 signatures, serous tubal intraepithelial carcinomas (STIC), and invasive HGSOC. Our findings reveal immune modulating mechanisms within precursor epithelium, characterized by chromosomal instability, persistent interferon (IFN) signaling, and dysregulated innate and adaptive immunity. FT precursors display elevated expression of MHC-class I, including HLA-E, and IFN-stimulated genes, typically linked to later-stage tumorigenesis. These molecular alterations coincide with progressive shifts in the tumor microenvironment, transitioning from immune surveillance in early STICs to immune suppression in advanced STICs and cancer. These insights identify potential biomarkers and therapeutic targets for HGSOC interception and clarify the molecular transitions from precancer to cancer.

Significance: Combined EZH2 and RAS pathway inhibitors kill KRAS-mutant colorectal cancer cells and promote durable tumor regression in vivo. These agents function by cooperatively suppressing the WNT pathway, driving differentiation, and epigenetically reprogramming cells to permit the induction of apoptotic signals, which then kill these more differentiated tumor cells., (©2024 The Authors; Published by the American Association for Cancer Research.)