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Understanding intratumoral heterogeneity—the molecular variation among cells within a tumor—promises to address outstanding questions in cancer biology and improve the diagnosis and treatment of specific cancer subtypes. Single-cell analyses, especially RNA sequencing and other genomics modalities, have been transformative in revealing novel biomarkers and molecular regulators associated with tumor growth, metastasis and drug resistance. However, these approaches fail to provide a complete picture of tumor biology, as information on cellular location within the tumor microenvironment is lost. New technologies leveraging multiplexed fluorescence, DNA, RNA and isotope labeling enable the detection of tens to thousands of cancer subclones or molecular biomarkers within their native spatial context. The expeditious growth in these techniques, along with methods for multiomics data integration, promises to yield a more comprehensive understanding of cell-to-cell variation within and between individual tumors. Here we provide the current state and future perspectives on the spatial technologies expected to drive the next generation of research and diagnostic and therapeutic strategies for cancer.This Review describes spatial omics and multiplexed imaging technologies and their current and future impact in studying tumor heterogeneity and cancer biology.

Tumor microenvironments (TMEs) contain vast amounts of information on patient’s cancer through their cellular composition and the spatial distribution of tumor cells and immune cell populations. Exploring variations in TMEs between patient groups, as well as determining the extent to which this information can predict outcomes such as patient survival or treatment success with emerging immunotherapies, is of great interest. Moreover, in the face of a large number of cell interactions to consider, we often wish to identify specific interactions that are useful in making such predictions. We present an approach to achieve these goals based on summarizing spatial relationships in the TME using spatial K functions, and then applying functional data analysis and random forest models to both predict outcomes of interest and identify important spatial relationships. This approach is shown to be effective in simulation experiments at both identifying important spatial interactions while also controlling the false discovery rate. We further used the proposed approach to interrogate two real data sets of Multiplexed Ion Beam Images of TMEs in triple negative breast cancer and lung cancer patients. The methods proposed are publicly available in a companion R package funkycells .

Contrary to the belief that basal-like breast cancers develop from mammary stem cells in BRCA1 mutation carriers, an aberrant luminal progenitor population might be the target for transformation in basal tumors in these individuals ( pages 842–844 ). Basal-like breast cancers arising in women carrying mutations in the BRCA1 gene, encoding the tumor suppressor protein BRCA1, are thought to develop from the mammary stem cell. To explore early cellular changes that occur in BRCA1 mutation carriers, we have prospectively isolated distinct epithelial subpopulations from normal mammary tissue and preneoplastic specimens from individuals heterozygous for a BRCA1 mutation. We describe three epithelial subsets including basal stem/progenitor, luminal progenitor and mature luminal cells. Unexpectedly, we found that breast tissue from BRCA1 mutation carriers harbors an expanded luminal progenitor population that shows factor-independent growth in vitro . Moreover, gene expression profiling revealed that breast tissue heterozygous for a BRCA1 mutation and basal breast tumors were more similar to normal luminal progenitor cells than any other subset, including the stem cell–enriched population. The c-KIT tyrosine kinase receptor (encoded by KIT ) emerged as a key marker of luminal progenitor cells and was more highly expressed in BRCA1 -associated preneoplastic tissue and tumors. Our findings suggest that an aberrant luminal progenitor population is a target for transformation in BRCA1 -associated basal tumors .

Spatial molecular data has transformed the study of disease microenvironments, though, larger datasets pose an analytics challenge prompting the direct adoption of single-cell RNA-sequencing tools including normalization methods. Here, we demonstrate that library size is associated with tissue structure and that normalizing these effects out using commonly applied scRNA-seq normalization methods will negatively affect spatial domain identification. Spatial data should not be specifically corrected for library size prior to analysis, and algorithms designed for scRNA-seq data should be adopted with caution.

Mammary stem cells The existence of mammary stem cells has been inferred from genetic analysis of human breast tissue, and from the fact that mouse mammary gland can regenerate from tissue fragments. Two groups now report the isolation of mammary stem cells from mice. Shackleton et al . use a technique based on the introduction of a marker for stem-cell function. A single cell from this population then showed its potency by regenerating an entire mammary gland in vivo . This population of cells is expanded in a pre-malignant mammary tumour model, providing support for the concept of a mammary cancer stem cell. In a paper published online, Stingl et al . report the use of a powerful limiting dilution transplant procedure to purify to near homogeneity a rare subset of adult mouse mammary cells that can individually regenerate an entire mammary gland within six weeks. The existence of mammary stem cells (MaSCs) has been postulated from evidence that the mammary gland can be regenerated by transplantation of epithelial fragments in mice 1 , 2 , 3 . Interest in MaSCs has been further stimulated by their potential role in breast tumorigenesis 4 . However, the identity and purification of MaSCs has proved elusive owing to the lack of defined markers. We isolated discrete populations of mouse mammary cells on the basis of cell-surface markers and identified a subpopulation (Lin - CD29 hi CD24 + ) that is highly enriched for MaSCs by transplantation. Here we show that a single cell, marked with a LacZ transgene, can reconstitute a complete mammary gland in vivo . The transplanted cell contributed to both the luminal and myoepithelial lineages and generated functional lobuloalveolar units during pregnancy. The self-renewing capacity of these cells was demonstrated by serial transplantation of clonal outgrowths. In support of a potential role for MaSCs in breast cancer, the stem-cell-enriched subpopulation was expanded in premalignant mammary tissue from MMTV- wnt-1 mice and contained a higher number of MaSCs. Our data establish that single cells within the Lin - CD29 hi CD24 + population are multipotent and self-renewing, properties that define them as MaSCs.

The transcription factor Gata-3 is a defining marker of the 'luminal' subtypes of breast cancer 1 , 2 , 3 , 4 . To gain insight into the role of Gata-3 in breast epithelial development and oncogenesis, we have explored its normal function within the mammary gland by conditionally deleting Gata-3 at different stages of development. We report that Gata-3 has essential roles in the morphogenesis of the mammary gland in both the embryo and adult. Through the discovery of a novel marker (β 3 -integrin) of luminal progenitor cells and their purification, we demonstrate that Gata-3 deficiency leads to an expansion of luminal progenitors and a concomitant block in differentiation. Remarkably, introduction of Gata-3 into a stem cell-enriched population induced maturation along the alveolar luminal lineage. These studies provide evidence for the existence of an epithelial hierarchy within the mammary gland and establish Gata-3 as a critical regulator of luminal differentiation.

The discovery of frequent 8p11-p12 amplifications in squamous cell lung cancer (SQLC) has fueled hopes that FGFR1, located inside this amplicon, might be a therapeutic target. In a clinical trial, only 11% of patients with 8p11 amplification (detected by FISH) responded to FGFR kinase inhibitor treatment. To understand the mechanism of FGFR1 dependency, we performed deep genomic characterization of 52 SQLCs with 8p11-p12 amplification, including 10 tumors obtained from patients who had been treated with FGFR inhibitors. We discovered somatically altered variants of FGFR1 with deletion of exons 1-8 that resulted from intragenic tail-to-tail rearrangements. These ectodomain-deficient FGFR1 variants (ΔEC-FGFR1) were expressed in the affected tumors and were tumorigenic in both in vitro and in vivo models of lung cancer. Mechanistically, breakage-fusion-bridges were the source of 8p11-p12 amplification, resulting from frequent head-to-head and tail-to-tail rearrangements. Generally, tail-to-tail rearrangements within or in close proximity upstream of FGFR1 were associated with FGFR1 dependency. Thus, the genomic events shaping the architecture of the 8p11-p12 amplicon provide a mechanistic explanation for the emergence of FGFR1-driven SQLC. Specifically, we believe that FGFR1 ectodomain-deficient and FGFR1-centered amplifications caused by tail-to-tail rearrangements are a novel somatic genomic event that might be predictive of therapeutically relevant FGFR1 dependency.

Genetic alterations in the fibroblast growth factor receptors (FGFRs) have been described in multiple solid tumours including bladder cancer, head and neck and lung squamous cell carcinoma (SqCC). However, recent clinical trials showed limited efficacy of FGFR-targeted therapy in lung SqCC, suggesting combination therapy may be necessary to improve patient outcomes. Here we demonstrate that FGFR therapy primes SqCC for cell death by increasing the expression of the pro-apoptotic protein BIM. We therefore hypothesised that combining BH3-mimetics, potent inhibitors of pro-survival proteins, with FGFR-targeted therapy may enhance the killing of SqCC cells. Using patient-derived xenografts and specific inhibitors of BCL-2, BCL-XL, and MCL-1, we identified a greater reliance of lung SqCC cells on BCL-XL and MCL-1 compared to BCL-2 for survival. However, neither BCL-XL nor MCL-1 inhibitors alone provided a survival benefit in combination FGFR therapy in vivo. Only triple BCL-XL, MCL-1, and FGFR inhibition resulted in tumour volume regression and prolonged survival in vivo, demonstrating the ability of BCL-XL and MCL-1 proteins to compensate for each other in lung SqCC. Our work therefore provides a rationale for the inhibition of MCL-1, BCL-XL, and FGFR1 to maximize therapeutic response in FGFR1 -expressing lung SqCC.

Inflammation is an important component of cancer diathesis and treatment-refractory inflammation is a feature of many chronic degenerative lung diseases. HSP90 is a 90kDa protein which functions as an ATP-dependent molecular chaperone that regulates the signalling conformation and expression of multiple protein client proteins especially oncogenic mediators. HSP90 inhibitors are in clinical development as cancer therapies but the myeleosuppressive and neutropenic effect of first generation geldanamycin-class inhibitors has confounded studies on the effects on HSP90 inhibitors on inflammation. To address this we assessed the ability of Ganetespib, a non-geldanamycin HSP90 blocker, to suppress lipopolysaccharide (LPS)-induced cellular infiltrates, proteases and inflammatory mediator and transcriptional profiles. Ganetespib (10-100 mg/kg, i.v.) did not directly cause myelosuppression, as assessed by video micrography and basal blood cell count, but it strongly and dose-dependently suppressed LPS-induced neutrophil mobilization into blood and neutrophil- and mononuclear cell-rich steroid-refractory lung inflammation. Ganetespib also suppressed B cell and NK cell accumulation, inflammatory cytokine and chemokine induction and MMP9 levels. These data identify non-myelosuppresssive HSP90 inhibitors as potential therapies for inflammatory diseases refractory to conventional therapy, in particular those of the lung.

Mammary stem cell control The ovarian hormones oestrogen and progesterone are involved in a complicated series of interactions in the mammary glands from the beginning of puberty to the menopause. Many of these changes are associated with cell proliferation, and breast cancer can result when errors occur. Two studies in this issue examine the effects of oestrogen and progesterone on mouse mammary stem cell (MaSC) function. They find that MaSC numbers decrease in virgin mice in the absence of both hormones due to ovariectomy or drug blockade, but increase with oestrogen and progesterone treatment. In addition, both groups implicate RANKL, a progesterone target known to be involved in bone remodelling and mammary gland formation, as an intermediary in the MaSC response to progesterone. The ovarian hormones oestrogen and progesterone increase breast cancer risk but the cellular mechanisms are unclear. Here it is shown that the size of the mammary stem cell pool in mice is regulated by steroid hormone signalling, although these cells lack the receptors for oestrogen and progesterone. The augmented pool could lead to clonal expansion of a mutated cell, possibly accounting for the increased incidence of breast cancer associated with pregnancy. The ovarian hormones oestrogen and progesterone profoundly influence breast cancer risk 1 , 2 , 3 , underpinning the benefit of endocrine therapies in the treatment of breast cancer 4 . Modulation of their effects through ovarian ablation or chemoprevention strategies also significantly decreases breast cancer incidence 5 , 6 . Conversely, there is an increased risk of breast cancer associated with pregnancy in the short term 7 . The cellular mechanisms underlying these observations, however, are poorly defined. Here we demonstrate that mouse mammary stem cells (MaSCs) 8 , 9 are highly responsive to steroid hormone signalling, despite lacking the oestrogen and progesterone receptors 10 . Ovariectomy markedly diminished MaSC number and outgrowth potential in vivo , whereas MaSC activity increased in mice treated with oestrogen plus progesterone. Notably, even three weeks of treatment with the aromatase inhibitor letrozole was sufficient to reduce the MaSC pool. In contrast, pregnancy led to a transient 11-fold increase in MaSC numbers, probably mediated through paracrine signalling from RANK ligand. The augmented MaSC pool indicates a cellular basis for the short-term increase in breast cancer incidence that accompanies pregnancy. These findings further indicate that breast cancer chemoprevention may be achieved, in part, through suppression of MaSC function.