Robust biomarker discovery through multiplatform multiplex image analysis of breast cancer clinical cohorts.

Spatial profiling of tissues promises to elucidate tumor-microenvironment interactions and enable development of spatial biomarkers to predict patient response to immunotherapy and other therapeutics. However, spatial biomarker discovery is often carried out on a single patient cohort or imaging technology, limiting statistical power and increasing the likelihood of technical artifacts. In order to analyze multiple patient cohorts profiled on different platforms, we developed methods for comparative data analysis from three disparate multiplex imaging technologies: 1) cyclic immunofluorescence data we generated from 102 breast cancer patients with clinical follow-up, in addition to publicly available 2) imaging mass cytometry and 3) multiplex ion-beam imaging data. We demonstrate similar single-cell phenotyping results across breast cancer patient cohorts imaged with these three technologies and identify cellular abundance and proximity-based biomarkers with prognostic value across platforms. In multiple platforms, we identified lymphocyte infiltration as independently associated with longer survival in triple negative and high-proliferation breast tumors. Then, a comparison of nine spatial analysis methods revealed robust spatial biomarkers. In estrogen receptor-positive disease, quiescent stromal cells close to tumor were more abundant in good prognosis tumors while tumor neighborhoods of mixed fibroblast phenotypes were enriched in poor prognosis tumors. In triple-negative breast cancer (TNBC), macrophage proximity to tumor and B cell proximity to T cells were greater in good prognosis tumors, while tumor neighborhoods of vimentin-positive fibroblasts were enriched in poor prognosis tumors. We also tested previously published spatial biomarkers in our ensemble cohort, reproducing the positive prognostic value of isolated lymphocytes and lymphocyte occupancy and failing to reproduce the prognostic value of tumor-immune mixing score in TNBC. In conclusion, we demonstrate assembly of larger clinical cohorts from diverse platforms to aid in prognostic spatial biomarker identification and validation.

The Complex Biology of the Obesity-Induced, Metastasis-Promoting Tumor Microenvironment in Breast Cancer.

Breast cancer is one of the most prevalent cancers in women contributing to cancer-related death in the advanced world. Apart from the menopausal status, the trigger for developing breast cancer may vary widely from race to lifestyle factors. Epidemiological studies refer to obesity-associated metabolic changes as a critical risk factor behind the progression of breast cancer. The plethora of signals arising due to obesity-induced changes in adipocytes present in breast tumor microenvironment, significantly affect the behavior of adjacent breast cells. Adipocytes from white adipose tissue are currently recognized as an active endocrine organ secreting different bioactive compounds. However, due to excess energy intake and increased fat accumulation, there are morphological followed by secretory changes in adipocytes, which make the breast microenvironment proinflammatory. This proinflammatory milieu not only increases the risk of breast cancer development through hormone conversion, but it also plays a role in breast cancer progression through the activation of effector proteins responsible for the biological phenomenon of metastasis. The aim of this review is to present a comprehensive picture of the complex biology of obesity-induced changes in white adipocytes and demonstrate the relationship between obesity and breast cancer progression to metastasis.

PPARgamma: A Potential Intrinsic and Extrinsic Molecular Target for Breast Cancer Therapy.

Over the last decades, the breast tumor microenvironment (TME) has been increasingly recognized as a key player in tumor development and progression and as a promising prognostic and therapeutic target for breast cancer patients. The breast TME, representing a complex network of cellular signaling-deriving from different stromal cell types as well as extracellular matrix components, extracellular vesicles, and soluble growth factors-establishes a crosstalk with cancer cells sustaining tumor progression. A significant emphasis derives from the tumor surrounding inflammation responsible for the failure of the immune system to effectively restrain breast cancer growth. Thus, effective therapeutic strategies require a deeper understanding of the interplay between tumor and stroma, aimed at targeting both the intrinsic neoplastic cells and the extrinsic surrounding stroma. In this scenario, peroxisome proliferator-activated receptor (PPAR) γ, primarily known as a metabolic regulator, emerged as a potential target for breast cancer treatment since it functions in breast cancer cells and several components of the breast TME. In particular, the activation of PPARγ by natural and synthetic ligands inhibits breast cancer cell growth, motility, and invasiveness. Moreover, activated PPARγ may educate altered stromal cells, counteracting the pro-inflammatory milieu that drive breast cancer progression. Interestingly, using Kaplan-Meier survival curves, PPARγ also emerges as a prognostically favorable factor in breast cancer patients. In this perspective, we briefly discuss the mechanisms by which PPARγ is implicated in tumor biology as well as in the complex regulatory networks within the breast TME. This may help to profile approaches that provide a simultaneous inhibition of epithelial cells and TME components, offering a more efficient way to treat breast cancer.

Activation of the S100A7/RAGE Pathway by IGF-1 Contributes to Angiogenesis in Breast Cancer.

BACKGROUND: Breast cancer (BC) mortality is increased among obese and diabetic patients. Both obesity and diabetes are associated with dysregulation of both the IGF-1R and the RAGE (Receptor for Advanced Glycation End Products) pathways, which contribute to complications of these disorders. The alarmin S100A7, signaling through the receptor RAGE, prompts angiogenesis, inflammation, and BC progression. METHODS: We performed bioinformatic analysis of BC gene expression datasets from published studies. We then used Estrogen Receptor (ER)-positive BC cells, CRISPR-mediated IGF-1R KO BC cells, and isogenic S100A7-transduced BC cells to investigate the role of IGF-1/IGF-1R in the regulation of S100A7 expression and tumor angiogenesis. To this aim, we also used gene silencing and pharmacological inhibitors, and we performed gene expression and promoter studies, western blotting analysis, ChIP and ELISA assays, endothelial cell proliferation and tube formation assay. RESULTS: S100A7 expression correlates with worse prognostic outcomes in human BCs. In BC cells, the IGF-1/IGF-1R signaling engages STAT3 activation and its recruitment to the S100A7 promoter toward S100A7 increase. In human vascular endothelial cells, S100A7 activates RAGE signaling and prompts angiogenic effects. CONCLUSIONS: In ER-positive BCs the IGF-1 dependent activation of the S100A7/RAGE signaling in adjacent endothelial cells may serve as a previously unidentified angiocrine effector. Targeting S100A7 may pave the way for a better control of BC, particularly in conditions of unopposed activation of the IGF-1/IGF-1R axis.

Gene expression of adipokines and adipokine receptors in the tumor microenvironment: associations of lower expression with more aggressive breast tumor features.

PURPOSE: Limited epidemiologic data are available on the expression of adipokines leptin (LEP) and adiponectin (ADIPOQ) and adipokine receptors (LEPR, ADIPOR1, ADIPOR2) in the breast tumor microenvironment (TME). The associations of gene expression of these biomarkers with tumor clinicopathology are not well understood. METHODS: NanoString multiplexed assays were used to assess the gene expression levels of LEP, LEPR, ADIPOQ, ADIPOR1, and ADIPOR2 within tumor tissues among 162 Black and 55 White women with newly diagnosed breast cancer. Multivariate mixed effects models were used to estimate associations of gene expression with breast tumor clinicopathology (overall and separately among Blacks). RESULTS: Black race was associated with lower gene expression of LEPR (P = 0.002) and ADIPOR1 (P = 0.01). Lower LEP, LEPR, and ADIPOQ gene expression were associated with higher tumor grade (P = 0.0007, P < 0.0001, and P < 0.0001, respectively) and larger tumor size (P < 0.0001, P = 0.0005, and P < 0.0001, respectively). Lower ADIPOQ expression was associated with ER- status (P = 0.0005), and HER2-enriched (HER2-E; P = 0.0003) and triple-negative (TN; P = 0.002) subtypes. Lower ADIPOR2 expression was associated with Ki67+ status (P = 0.0002), ER- status (P < 0.0001), PR- status (P < 0.0001), and TN subtype (P = 0.0002). Associations of lower adipokine and adipokine receptor gene expression with ER-, HER2-E, and TN subtypes were confirmed using data from The Cancer Genome Atlas (P-values < 0.005). CONCLUSION: These findings suggest that lower expression of ADIPOQ, ADIPOR2, LEP, and LEPR in the breast TME might be indicators of more aggressive breast cancer phenotypes. Validation of these findings are warranted to elucidate the role of the adipokines and adipokine receptors in long-term breast cancer prognosis.

Cancer associated fibroblasts: role in breast cancer and potential as therapeutic targets.

Introduction: Cancer associated fibroblasts (CAFs) are the largest population of stromal cells in breast tumors. Emerging evidence has suggested that CAFs are important players not only in fostering tumor growth and spread but also in altering the tumor response to therapeutic agents. On the basis of these observations, huge efforts have been made to exploit CAFs as potential targets for breast cancer therapy.Areas covered: The current understanding of the hallmarks and biology of CAFs, their multilayered interplay with various cell populations of breast tumor microenvironment toward cancer initiation, progression, metastasis and resistance to anticancer therapies are discussed. In addition, a comprehensive overview of the CAFs-based molecular druggable targets in breast tumors is provided. The most relevant literature, in particular the studies retrieved in Medline in the last 10 years, served for this purpose.Expert opinion: The interest on CAFs as a target to fight breast cancer has becoming a hot topic for drug discovery. Indeed, several CAFs-targeted approaches are emerging as appealing therapeutic strategies in breast cancer. At pre-clinical level, this research field is speedily advancing toward the assessment of successful tactics targeting CAFs in breast cancer. Therefore, anti-CAFs therapies may display an intriguing potential to be exploited in clinical studies.

Modulating Tumor-Associated Macrophage Polarization by Synthetic and Natural PPARγ Ligands as a Potential Target in Breast Cancer.

Activation of peroxisome proliferator-activated receptor gamma (PPARγ) elicits anti-proliferative effects on different tumor cells, including those derived from breast cancer. PPARγ is also expressed in several cells of the breast tumor microenvironment, among which tumor associated macrophages (TAMs) play a pivotal role in tumor progression and metastasis. We explored the ability of synthetic and natural PPARγ ligands to modulate TAM polarization. The ligands included rosiglitazone (BRL-49653), and two docosahexaenoic acid (DHA) conjugates, N-docosahexaenoyl ethanolamine (DHEA) and N-docosahexaenoyl serotonin (DHA-5-HT). Human THP-1 monocytic cells were differentiated into M0, M1 and M2 macrophages that were characterized by qRT-PCR, ELISA and western blotting. A TAM-like phenotypic state was generated by adding two different breast cancer cell conditioned media (BCC-CM) to the cultures. Macrophages exposed to BCC-CM concomitantly exhibited M1 and M2 phenotypes. Interestingly, rosiglitazone, DHEA and DHA-5-HT attenuated cytokine secretion by TAMs, and this effect was reversed by the PPARγ antagonist GW9662. Given the key role played by PPARγ in the crosstalk between cancer cells and TAMs in tumor progression, its activation via endogenous or synthetic ligands may lead to novel strategies that target both epithelial neoplastic cells and the tumor microenvironment.

CD4+ and CD8+ T cells have opposing roles in breast cancer progression and outcome.

The Cancer Immunoediting concept has provided critical insights suggesting dual functions of immune system during the cancer initiation and development. However, the dynamics and roles of CD4+ and CD8+ T cells in the pathogenesis of breast cancer remain unclear. Here we utilized two murine breast cancer models (4T1 and E0771) and demonstrated that both CD4+ and CD8+ T cells were increased and involved in immune responses, but with distinct dynamic trends in breast cancer development. In addition to cell number increases, CD4+ T cells changed their dominant subsets from Th1 in the early stages to Treg and Th17 cells in the late stages of the cancer progression. We also analyzed CD4+ and CD8+ T cell infiltration in primary breast cancer tissues from cancer patients. We observed that CD8+ T cells are the key effector cell population mediating effective anti-tumor immunity resulting in better clinical outcomes. In contrast, intra-tumoral CD4+ T cells have negative prognostic effects on breast cancer patient outcomes. These studies indicate that CD4+ and CD8+ T cells have opposing roles in breast cancer progression and outcomes, which provides new insights relevant for the development of effective cancer immunotherapeutic approaches.