High-throughput surface marker screen on primary human breast tissues reveals further cellular heterogeneity

Background Normal human breast tissues are a heterogeneous mix of epithelial and stromal subtypes in different cell states. Delineating the spectrum of cellular heterogeneity will provide new insights into normal cellular properties within the breast tissue that might become dysregulated in the initial stages of cancer. Investigation of surface marker expression provides a valuable approach to resolve complex cell populations. However, the majority of cell surface maker expression of primary breast cells have not been investigated. Methods To determine the differences in expression of a range of uninvestigated cell surface markers between the normal breast cell subpopulations, primary human breast cells were analysed using high-throughput flow cytometry for the expression of 242 cell surface proteins in conjunction with EpCAM/CD49f staining. Results We identified 35 surface marker proteins expressed on normal breast epithelial and/or stromal subpopulations that were previously unreported. We also show multiple markers were equally expressed in all cell populations (e.g. CD9, CD59, CD164) while other surface markers were confirmed to be enriched in different cell lineages: CD24, CD227 and CD340 in the luminal compartment, CD10 and CD90 in the basal population, and CD34 and CD140b on stromal cells. Conclusions Our dataset of CD marker expression in the normal breast provides better definition for breast cellular heterogeneity.

Quantifying Breast Cancer-Driven Fiber Alignment and Collagen Deposition in Primary Human Breast Tissue

Solid tumor progression is significantly influenced by interactions between cancer cells and the surrounding extracellular matrix (ECM). Specifically, the cancer cell-driven changes to ECM fiber alignment and collagen deposition impact tumor growth and metastasis. Current methods of quantifying these processes are incomplete, require simple or artificial matrixes, rely on uncommon imaging techniques, preclude the use of biological and technical replicates, require destruction of the tissue, or are prone to segmentation errors. We present a set of methodological solutions to these shortcomings that were developed to quantify these processes in cultured, ex vivo human breast tissue under the influence of breast cancer cells and allow for the study of ECM in primary breast tumors. Herein, we describe a method of quantifying fiber alignment that can analyze complex native ECM from scanning electron micrographs that does not preclude the use of replicates and a high-throughput mechanism of quantifying collagen content that is non-destructive. The use of these methods accurately recapitulated cancer cell-driven changes in fiber alignment and collagen deposition observed by visual inspection. Additionally, these methods successfully identified increased fiber alignment in primary human breast tumors when compared to human breast tissue and increased collagen deposition in lobular breast cancer when compared to ductal breast cancer. The successful quantification of fiber alignment and collagen deposition using these methods encourages their use for future studies of ECM dysregulation in human solid tumors.

RNAi Screening in Primary Human Breast Biopsies Identifies Mitochondrial Glutamate Flux as A Metabolic Vulnerability in Locally Advanced Breast Cancer

Background: Breast cancer (BC) is a leading cause of death in women[1]. Women with Locally Advanced Breast Cancer (LABC) have high risk disease with either large primary breast tumours and/or lymph node involvement. While neoadjuvant chemotherapy eradicates breast cancer in approximately one-third of cases prior to surgery, almost 70% of patients have residual disease and many will require additional chemotherapy post-operatively. Improving pre-operative efficacy of neoadjuvant systemic treatments while reducing their iatrogenicities are critical unmet needs. Methods: Here, we develop an RNA interference (RNAi) screening approach using conditionally reprogrammed primary LABC biopsies to identify genes of the mitochondrial Solute Ligand Carrier 25 (SLC25) family that support LABC cell viability. Results: We report that silencing SLC25A12, -A15, and -A18 genes, involved in glutamate and ornithine flux, augment 5-fluorouracil (5FU) cytotoxic effectiveness in LABC cells. Conclusions: Our data suggest glutamate metabolism may be a tumour-specific metabolic vulnerability in LABC. Furthermore, we demonstrate that RNAi screening in conditionally reprogrammed primary human breast cells can identify novel targets for the development of non-genotoxic BC treatments.

Phenotypic functional activities of monocyte change during crosstalk with breast cancer cell and enhancing effect of metformin of IFN-γ-associated antitumor cytokine production

BackgroundImmune activities of monocytes (MOs) can be altered within the microenvironment of solid malignancies, including breast cancer. Metformin (1,1-dimethylbiguanide hydrochloride, MET), has been shown to decrease tumor cell proliferation, but its effects have yet to be explored with respect to the crosstalk between monocytes and breast cancer cells. Here, we investigated the effects of MET on overall phenotypic functional activities of autologous MOs during the interplay with primary breast cancer cells.MethodsHuman primary breast cancer cells were either cultured alone or co-cultured with autologous MOs before treatment with MET.ResultsMET downregulated both breast cancer cell proliferation and the ratio of phosphorylated Akt (p-Akt)-to-Akt in breast cancer cells. Additionally, we observed that, in the absence of MET treatment, the levels of LDH-based cytotoxicity, catalase, intracellular free calcium ions (ifCa2+), IL-10 and arginase activity were significantly reduced in co-cultures compared to those of MOs cultivated alone whereas levels of iNOS were significantly increased (for all comparisons, p < 0.05). In contrast, MET upregulated breast cancer cell LDH-based cytotoxicity levels when co-cultured with MO. MET also induced upregulation of both the inducible enzymatic activity of nitric oxide synthase (iNOS) and arginase activity in MO cells and co-culture systems, although these differences did not reach significant levels for iNOS activity (p > 0.05). MET greatly decreased phagocytic activity in isolated MOs while inducing a robust increase of catalase activity in co-culture systems and of superoxide dismutase (SOD) activity in MOs, but not in MOs co-cultured with breast cancer cells. MET strongly upregulated the levels of ifCa2+ in co-culture systems and IFN-γ production in both isolated MOs and co-culture systems. Moreover, MET treatment markedly downregulated IL-10 production in MOs, while inducing a slight increase in co-cultures (p > 0.05).ConclusionsOur results show that the phenotypic functional activities of MOs change when co-cultured with primary human breast cancer cells. Furthermore, treatment with MET induced enhancing effects on the production of antitumor cytokine IFN-γ and ifCa2+, as well as cytotoxicity during breast cancer cell-MO crosstalk.Novel Highlights includeFirst analysis of the anti-tumoral effects of Metformin on primary human breast cancer cells and the crosstalk with autologous monocytes.Phenotypic functional activities of monocytes change during their interplay with breast cancer cells, which is improved by upregulation of IFN-γ after Metformin treatment.Metformin induces downregulation of phosphorylated-Akt1/2-to-Akt1/2 ratio in breast cancer cells.Metformin downregulates phagocytic capacity of monocyte from breast cancer patients.

Characterization of organoid cultured human breast cancer

Organoid cultures are increasingly used to model human cancers experimentally with a view to tailoring personalized medicine and predicting drug responses. Breast cancer is no exception, but in particular, primary breast cancer poses some inherent difficulties due to the frequent presence of residual non-malignant cells in the biopsies. We originally developed an assay for the distinction between malignant and non-malignant structures in primary breast cancer organoid cultures (Petersen et al., Proc Natl Acad Sci (USA) 89(19):9064–8, 1992). Here, we apply this assay to assess the frequency of normal-like organoids in primary breast carcinoma cultures and the cellular composition as a consequence of passaging. We find that in consecutively collected samples of primary human breast cancers, residual non-malignant tissues were observed histologically in five out of ten biopsies. Based on relevant morphogenesis and correct polarization as recorded by expression in luminal epithelial cells of mucin 1 (Muc1), occludin, and keratin 19 (K19) and expression in basal cells of integrin β4, p63, and K14, non-malignant organoids were present in all primary human breast cancer-derived cultures. Furthermore, passaging in a contemporary culture medium was in favor of the selective expansion of basal-like cells. We conclude that organoid cultures of human breast cancers are most representative of the tissue origin in primary culture.