Tumor Cell Spatial Organization Directs EGFR/RAS/RAF Pathway Primary Therapy Resistance through YAP Signaling.

Non-small cell lung cancers (NSCLC) harboring common mutations in EGFR and KRAS characteristically respond transiently to targeted therapies against those mutations, but invariably, tumors recur and progress. Resistance often emerges through mutations in the therapeutic target or activation of alternative signaling pathways. Mechanisms of acute tumor cell resistance to initial EGFR (EGFRi) or KRASG12C (G12Ci) pathway inhibition remain poorly understood. Our study reveals that acute response to EGFR/RAS/RAF-pathway inhibition is spatial and culture context specific. In vivo, EGFR mutant tumor xenografts shrink by > 90% following acute EGFRi therapy, and residual tumor cells are associated with dense stroma and have increased nuclear YAP. Interestingly, in vitro EGFRi induced cell cycle arrest in NSCLC cells grown in monolayer, while 3D spheroids preferentially die upon inhibitor treatment. We find differential YAP nuclear localization and activity, driven by the distinct culture conditions, as a common resistance mechanism for selective EGFR/KRAS/BRAF pathway therapies. Forced expression of the YAPS127A mutant partially protects cells from EGFR-mediated cell death in spheroid culture. These studies identify YAP activation in monolayer culture as a non-genetic mechanism of acute EGFR/KRAS/BRAF therapy resistance, highlighting that monolayer vs spheroid cell culture systems can model distinct stages of patient cancer progression.

Tumor-associated macrophages restrict CD8+ T cell function through collagen deposition and metabolic reprogramming of the breast cancer microenvironment.

Tumor progression is accompanied by fibrosis, a condition of excessive extracellular matrix accumulation, which is associated with diminished antitumor immune infiltration. Here we demonstrate that tumor-associated macrophages (TAMs) respond to the stiffened fibrotic tumor microenvironment (TME) by initiating a collagen biosynthesis program directed by transforming growth factor-ß. A collateral effect of this programming is an untenable metabolic milieu for productive CD8+ T cell antitumor responses, as collagen-synthesizing macrophages consume environmental arginine, synthesize proline and secrete ornithine that compromises CD8+ T cell function in female breast cancer. Thus, a stiff and fibrotic TME may impede antitumor immunity not only by direct physical exclusion of CD8+ T cells but also through secondary effects of a mechano-metabolic programming of TAMs, which creates an inhospitable metabolic milieu for CD8+ T cells to respond to anticancer immunotherapies.

Mechanosensitive hormone signaling promotes mammary progenitor expansion and breast cancer risk.

Tissue stem-progenitor cell frequency has been implicated in tumor risk and progression, but tissue-specific factors linking these associations remain ill-defined. We observed that stiff breast tissue from women with high mammographic density, who exhibit increased lifetime risk for breast cancer, associates with abundant stem-progenitor epithelial cells. Using genetically engineered mouse models of elevated integrin mechanosignaling and collagen density, syngeneic manipulations, and spheroid models, we determined that a stiff matrix and high mechanosignaling increase mammary epithelial stem-progenitor cell frequency and enhance tumor initiation in vivo. Augmented tissue mechanics expand stemness by potentiating extracellular signal-related kinase (ERK) activity to foster progesterone receptor-dependent RANK signaling. Consistently, we detected elevated phosphorylated ERK and progesterone receptors and increased levels of RANK signaling in stiff breast tissue from women with high mammographic density. The findings link fibrosis and mechanosignaling to stem-progenitor cell frequency and breast cancer risk and causally implicate epidermal growth factor receptor-ERK-dependent hormone signaling in this phenotype.

A convolutional neural network STIFMap reveals associations between stromal stiffness and EMT in breast cancer.

Intratumor heterogeneity associates with poor patient outcome. Stromal stiffening also accompanies cancer. Whether cancers demonstrate stiffness heterogeneity, and if this is linked to tumor cell heterogeneity remains unclear. We developed a method to measure the stiffness heterogeneity in human breast tumors that quantifies the stromal stiffness each cell experiences and permits visual registration with biomarkers of tumor progression. We present Spatially Transformed Inferential Force Map (STIFMap) which exploits computer vision to precisely automate atomic force microscopy (AFM) indentation combined with a trained convolutional neural network to predict stromal elasticity with micron-resolution using collagen morphological features and ground truth AFM data. We registered high-elasticity regions within human breast tumors colocalizing with markers of mechanical activation and an epithelial-to-mesenchymal transition (EMT). The findings highlight the utility of STIFMap to assess mechanical heterogeneity of human tumors across length scales from single cells to whole tissues and implicates stromal stiffness in tumor cell heterogeneity.

Mechanostimulation of breast myoepithelial cells induces functional changes associated with DCIS progression to invasion.

Women with ductal carcinoma in situ (DCIS) have an increased risk of progression to invasive breast cancer. Although not all women with DCIS will progress to invasion, all are treated as such, emphasising the need to identify prognostic biomarkers. We have previously shown that altered myoepithelial cells in DCIS predict disease progression and recurrence. By analysing DCIS duct size in sections of human breast tumour samples, we identified an associated upregulation of integrin ß6 and an increase in periductal fibronectin deposition with increased DCIS duct size that associated with the progression of DCIS to invasion. Our modelling of the mechanical stretching myoepithelial cells undergo during DCIS progression confirmed the upregulation of integrin ß6 and fibronectin expression in isolated primary and cell line models of normal myoepithelial cells. Our studies reveal that this mechanostimulated DCIS myoepithelial cell phenotype enhances invasion in a TGFß-mediated upregulation of MMP13. Immunohistochemical analysis identified that MMP13 was specifically upregulated in DCIS, and it was associated with progression to invasion. These findings implicate tissue mechanics in altering the myoepithelial cell phenotype in DCIS, and that these alterations may be used to stratify DCIS patients into low and high risk for invasive progression.

Immunosuppressive glycoproteins associate with breast tumor fibrosis and aggression.

Tumors feature elevated sialoglycoprotein content. Sialoglycoproteins promote tumor progression and are linked to immune suppression via the sialic acid-Siglec axis. Understanding factors that increase sialoglycoprotein biosynthesis in tumors could identify approaches to improve patient response to immunotherapy. We quantified higher levels of sialoglycoproteins in the fibrotic regions within human breast tumor tissues. Human breast tumor subtypes, which are more fibrotic, similarly featured increased sialoglycoprotein content. Further analysis revealed the breast cancer cells as the primary cell type synthesizing and secreting the tumor tissue sialoglycoproteins and confirmed that the more aggressive, fibrotic breast cancer subtypes expressed the highest levels of sialoglycoprotein biosynthetic genes. The more aggressive breast cancer subtypes also featured greater infiltration of immunosuppressive SIGLEC7, SIGLEC9, and SIGLEC10-pos myeloid cells, indicating that triple-negative breast tumors had higher expression of both immunosuppressive Siglec receptors and their cognate ligands. The findings link sialoglycoprotein biosynthesis and secretion to tumor fibrosis and aggression in human breast tumors. The data suggest targeting of the sialic acid-Siglec axis may comprise an attractive therapeutic target particularly for the more aggressive HER2+ and triple-negative breast cancer subtypes.

Tissue mechanics in stem cell fate, development, and cancer.

Cells in tissues experience a plethora of forces that regulate their fate and modulate development and homeostasis. Cells sense mechanical cues through localized mechanoreceptors or by influencing cytoskeletal or plasma membrane organization. Cells translate force and modulate their behavior through a process termed mechanotransduction. Cells tune their tension upon exposure to chronic force by engaging cellular machinery that modulates actin tension, which in turn stimulates matrix remodeling and stiffening and alters cell-cell adhesions until cells achieve a state of tensional homeostasis. Loss of tensional homeostasis can be induced through oncogene activity and/or tissue fibrosis, accompanies tumor progression, and is associated with increased cancer risk. The mechanical stresses that develop in tumors can also foster the mesenchymal-like transdifferentiation of cells to induce a stem-like phenotype that contributes to their aggression, metastatic dissemination, and treatment resistance. Thus, strategies that ameliorate tumor mechanics may comprise an effective strategy to prevent aggressive tumor behavior.

Improving DCIS diagnosis and predictive outcome by applying artificial intelligence.

Breast ductal carcinoma in situ (DCIS) is a preinvasive lesion that is considered to be a precursor to invasive breast cancer. Nevertheless, not all DCIS will progress to invasion. Current histopathological classification systems are unable to predict which cases will or will not progress, and therefore many women with DCIS may be overtreated. Artificial intelligence (AI) image-based analysis methods have potential to identify and analyze novel features that may facilitate tumor identification, prediction of disease outcome and response to treatment. Indeed, these methods prove promising for accurately identifying DCIS lesions, and show potential clinical utility in the therapeutic stratification of DCIS patients. Here, we review how AI techniques in histopathology may aid diagnosis and clinical decisions in regards to DCIS, and how such techniques could be incorporated into clinical practice.

Derivation of a nuclear heterogeneity image index to grade DCIS.

Abnormalities in cell nuclear morphology are a hallmark of cancer. Histological assessment of cell nuclear morphology is frequently used by pathologists to grade ductal carcinoma in situ (DCIS). Objective methods that allow standardization and reproducibility of cell nuclear morphology assessment have potential to improve the criteria needed to predict DCIS progression and recurrence. Aggressive cancers are highly heterogeneous. We asked whether cell nuclear morphology heterogeneity could be incorporated into a metric to classify DCIS. We developed a nuclear heterogeneity image index to objectively, and quantitatively grade DCIS. A whole-tissue cell nuclear morphological analysis, that classified tumors by the worst ten percent in a duct-by-duct manner, identified nuclear size ranges associated with each DCIS grade. Digital image analysis further revealed increasing heterogeneity within ducts or between ducts in tissues of worsening DCIS grade. The findings illustrate how digital image analysis comprises a supplemental tool for pathologists to objectively classify DCIS and in the future, may provide a method to predict patient outcome through analysis of nuclear heterogeneity.

Stiff stroma increases breast cancer risk by inducing the oncogene ZNF217.

Women with dense breasts have an increased lifetime risk of malignancy that has been attributed to a higher epithelial density. Quantitative proteomics, collagen analysis, and mechanical measurements in normal tissue revealed that stroma in the high-density breast contains more oriented, fibrillar collagen that is stiffer and correlates with higher epithelial cell density. microRNA (miR) profiling of breast tissue identified miR-203 as a matrix stiffness-repressed transcript that is downregulated by collagen density and reduced in the breast epithelium of women with high mammographic density. Culture studies demonstrated that ZNF217 mediates a matrix stiffness- and collagen density-induced increase in Akt activity and mammary epithelial cell proliferation. Manipulation of the epithelium in a mouse model of mammographic density supported a causal relationship between stromal stiffness, reduced miR-203, higher levels of the murine homolog Zfp217, and increased Akt activity and mammary epithelial proliferation. ZNF217 was also increased in the normal breast epithelium of women with high mammographic density, correlated positively with epithelial proliferation and density, and inversely with miR-203. The findings identify ZNF217 as a potential target toward which preexisting therapies, such as the Akt inhibitor triciribine, could be used as a chemopreventive agent to reduce cancer risk in women with high mammographic density.

Fibrosis and cancer: A strained relationship.

Tumors are characterized by extracellular matrix (ECM) deposition, remodeling, and cross-linking that drive fibrosis to stiffen the stroma and promote malignancy. The stiffened stroma enhances tumor cell growth, survival and migration and drives a mesenchymal transition. A stiff ECM also induces angiogenesis, hypoxia and compromises anti-tumor immunity. Not surprisingly, tumor aggression and poor patient prognosis correlate with degree of tissue fibrosis and level of stromal stiffness. In this review, we discuss the reciprocal interplay between tumor cells, cancer associated fibroblasts (CAF), immune cells and ECM stiffness in malignant transformation and cancer aggression. We discuss CAF heterogeneity and describe its impact on tumor development and aggression focusing on the role of CAFs in engineering the fibrotic tumor stroma and tuning tumor cell tension and modulating the immune response. To illustrate the role of mechanoreciprocity in tumor evolution we summarize data from breast cancer and pancreatic ductal carcinoma (PDAC) studies, and finish by discussing emerging anti-fibrotic strategies aimed at treating cancer.