Characterisation of the Stromal Microenvironment in Lobular Breast Cancer

SIMPLE SUMMARY: Invasive lobular breast cancer (ILC) accounts for approximately 5–15% of breast cancers, and although response rates to treatments are initially good, an ILC diagnosis is associated with adverse long-term outcomes; better treatments, specifically targeted to this sub-type of breast cancer, are required to improve patient survival. The tumor microenvironment (TME) plays an important role in determining how cancers respond to treatment, and in this study, we carried out an in-depth analysis of the TME in ILC following laser-capture microdissection of the tumor stroma, and analysis of primary cancer-associated fibroblasts (CAFs), which comprise the majority of non-malignant cells within tumors. This identified changes in genes involved in regulation of the extracellular matrix and also growth factor signaling pathways that were differentially regulated in ILC. Further analysis of breast cancer datasets showed that two of these genes which encode a secreted metalloproteinase (PAPPA) and a metalloproteinase inhibitor (TIMP2) were associated with survival outcomes in ILC. ABSTRACT: Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer, and it exhibits a number of clinico-pathological characteristics distinct from the more common invasive ductal carcinoma (IDC). We set out to identify alterations in the tumor microenvironment (TME) of ILC. We used laser-capture microdissection to separate tumor epithelium from stroma in 23 ER+ ILC primary tumors. Gene expression analysis identified 45 genes involved in regulation of the extracellular matrix (ECM) that were enriched in the non-immune stroma of ILC, but not in non-immune stroma from ER+ IDC or normal breast. Of these, 10 were expressed in cancer-associated fibroblasts (CAFs) and were increased in ILC compared to IDC in bulk gene expression datasets, with PAPPA and TIMP2 being associated with better survival in ILC but not IDC. PAPPA, a gene involved in IGF-1 signaling, was the most enriched in the stroma compared to the tumor epithelial compartment in ILC. Analysis of PAPPA- and IGF1-associated genes identified a paracrine signaling pathway, and active PAPP-A was shown to be secreted from primary CAFs. This is the first study to demonstrate molecular differences in the TME between ILC and IDC identifying differences in matrix organization and growth factor signaling pathways

The role of stromal-derived IGF1 signalling in invasive lobular carcinoma

Invasive lobular breast carcinoma (ILC) is the second most frequently diagnosed invasive breast cancer subtype. Besides E-cadherin loss, the prominent genetic hallmark of ILC, this subtype is characterised by unique pathologic, molecular, and clinical features, which differ from the other breast cancer subtypes. Recent studies have shown that loss of E-Cadherin, the hallmark of the ILC, sensitises the tumour cells to insulin-like growth factor 1 (IGF-1) signalling. However, the involvement of such activation in ILC tumourigenesis and metastasis remains unclear. Here we aim to study this pathway further in human and mouse models of ILC and in a large cohort of patients with primary operable breast cancer with mature follow-up. We initially characterised the pathway using quantitative RT-PCR, where significant expression of Igf1 was found in mouse ILC-derived cancer-associated fibroblasts (CAF), with no measurable expression in the mouse epithelial tumour cells. Both enzyme-linked immunosorbent assays (ELISA) and Forward phase protein arrays (FPPA) confirmed stromal secretion of IGF1 from the tumour cell-derived CAFs. Moreover, IGF-1 receptor stimulation in ILC tumour cells in response to recombinant IGF-1 or CAF-derived conditioned medium was observed. Such activation was successfully blocked upon treatment with an IGF1 receptor inhibitor. Additionally, the effect of IGF-1 on human and mouse ILC cell proliferation was assessed in vitro, where we showed that both recombinant IGF-1 and CAF-derived conditioned medium resulted in the increased proliferation rate of the ILC tumour cells in comparison with the control cells. In contrast, adding an IGF-1 receptor inhibitor strongly reduced their proliferation. As the role of the tumour microenvironment in tumour initiation and progression is increasingly recognised, an in vivo study of the ILC mouse model was performed. We injected ILC epithelial cells alone or with CAFs into mice. The co-injection with CAFs significantly accelerated tumour growth compared to mice injected with epithelial cells alone. Furthermore, significantly increased expression of Ki67 (proliferative marker) and CD31 (endothelial cell marker) was observed in the tumours of the co-injected mice. This project also aimed to clarify the clinical significance of IGF-1 signalling in human breast cancer. We carried out immunohistochemical analysis in a large cohort of 850 samples of various breast cancer subtypes (lobular, ductal and others) for E-Cadherin, IGF-1 receptor and other members of the IGF-1 signalling pathway. These studies revealed differential patterns of expression of these proteins and highlighted their significant association with cancer-specific survival as well as with the other clinicopathological features of the patients. To conclude, this work could provide deep insights into the importance of tumour-stromal IGF-1 signalling in breast cancer, particularly ILC tumourigenesis and metastasis. It will also help improve subsequent investigations and select patients for future targeting of IGF-1 signalling