Jonathan A. R. Gordon, Mark F. Evans, Prachi N. Ghule, Kyra Lee, Pamela Vacek, Brian L. Sprague, Donald L. Weaver, Gary S. Stein, Janet L. Stein
Abstract
The tumor microenvironment is a complex mixture of cell types that bi-directionally interact and influence tumor initiation, progression, recurrence, and patient survival. Mesenchymal stromal cells (MSCs) of the tumor microenvironment engage in crosstalk with cancer cells to mediate epigenetic control of gene expression. We identified CD90+ MSCs residing in the tumor microenvironment of patients with invasive breast cancer that exhibit a unique gene expression signature. Single-cell transcriptional analysis of these MSCs in tumor-associated stroma identified a distinct subpopulation characterized by increased expression of genes functionally related to extracellular matrix signaling. Blocking the TGFβ pathway reveals that these cells directly contribute to cancer cell proliferation. Our findings provide novel insight into communication between breast cancer cells and MSCs that are consistent with an epithelial to mesenchymal transition and acquisition of competency for compromised control of proliferation, mobility, motility, and phenotype.
Introduction
Mesenchymal stromal/stem-like cells (MSC) are a heterogeneous population of cells that reside primarily in bone marrow and perivascular regions, but can also be found in limited quantities in adipose, muscle and other tissues, as well as circulating in peripheral blood [1]. The canonical role of MSCs is to regenerate and repair adult tissues through their multipotent capacity to differentiate into osteoblasts, adipocytes, chondrocytes, myoblasts, and in therapeutic applications, into cardiac myocytes and neurons [2]. Even though these cells provide a vital function, the steady state amount of MSCs in the bone marrow is relatively low compared to mononuclear and hematopoietic cells, ranging from 0.1–1 MSC per 10,000 cells [3]. In this capacity, MSCs are vital in their distinct ability to repair damaged tissues. These cells also have other regulatory properties including modulation of immune response [4], stimulation of capillary formation [5], regulation of hematopoietic stem cells [6], paracrine signaling through cytokine production [7, 8], as well as modulation of cancer cell activity [9–13].
Materials and methods
Isolation of MSCs from breast cancer patients
Patient-derived MSCs were obtained from fresh breast tumor specimens from patients undergoing surgery at University of Vermont Medical Center. Tumor tissues were washed with PBS, cut into small pieces, and digested with 3 mg/mL collagenase I (Sigma) and 5 MU/mL of DNase I (Calbiochem) in PBS for 2 hours at 37°C. Cells were passed through a 70-μm strainer filter and negatively selected for CD45 expression using CD45 Microbeads (Miltenyi Biotec) and separated using magnetic cell separation LD columns (Miltenyi Biotec). The remaining cells were positively selected by treating with CD90 Microbeads and then selected using an MS column (Miltenyi Biotec). Cells were collected by centrifugation and then frozen (-150°C) in Bambanker until used directly for experiments
Results
Stroma from breast cancer patients contain CD90+ MSCs
For this study we utilized a multifaceted sampling approach to evaluate gene expression signatures related to MSCs residing in the breast stroma of patients with screen detected invasive breast cancer, symptom detected breast cancer and DCIS (Fig 1). A small amount of breast tissue (~4 mm3, 0.019–0.025 g) was sampled from 15 individual patients undergoing resection of screen detected invasive breast tumors. From each patient two individual regions were sampled by biopsy punch, one region distal from the pathologically identified tumor (referred to as “patient normal stroma” (PNS)) and one region directly adjacent and/or containing tumor tissue (referred to as “patient tumor stroma” (PTS)).
Discussion
The tumor microenvironment plays a distinct role in all aspects of tumorigenesis, including the establishment of oncogenic character, maintenance and growth of the malignant tumor and malignant/metastatic progression. It is well established that within the tumor microenvironment cells of the immune system, such as macrophages, neutrophils, mast cells, myeloid-derived suppressor cells (MDSCs), dendritic cells (DCs), and natural killer (NK) cells and those of adaptive immunity (T and B lymphocytes), play a complex role in the tumor niche. Normally, in cases of an invading pathogen, these cells would act to initiate an inflammatory response; however in the tumor microenvironment, these cells are frequently dysregulated or functionally impaired, which in some cases can promote cancer progression through extracellular signaling effectors and recruit or modify cells in the surrounding environment [37]. A significant portion of the surrounding environment is composed of tumor stromal cells of mesenchymal origin (mesenchymal stromal cells, fibroblasts, endothelial cells, and pericytes). Interactions that occur between immune cells and cells of the tumor itself are highly complex, and involve numerous cytokines, chemokines, and mitogens that directly affect tumor progression and outcomes.
Conclusion
Our results suggest that TGFβ signaling may be more important in driving pro-tumor effects from cancer-associated MSCs. Several potential anti-TGFβ inhibitors are currently under clinical development in phase I/II trials to treat primary tumors [86], but others have relatively poor success in clinical cancer treatment [87]. Patients with primary tumor itself may respond to anti-TGFβ therapies, but patients with cancer-associated MSCs may benefit from a prophylactic or targeted treatment with TGFβ inhibitor to revert activated tumor-associated MSCs to a naïve state. Our findings provide novel insight into communication between breast cancer cells and MSCs that are consistent with acquisition of competency for compromised control of proliferation, mobility, motility, and phenotype.
Acknowledgments
The authors thank Scott Tighe and Jess Hoffman for support with Next-Generation Sequencing including 10X Chromium single cell sorting/sequencing performed in the Vermont Integrative Genomics Resource Massively Parallel Sequencing Facility. The authors also thank Dr. Roxana del Rio-Guerra for assistance with cell sorting performed in the Harry Hood Bassett Flow Cytometry and Cell Sorting (FCCS) Facility at UVM Larner College of Medicine.
Citation: Gordon JAR, Evans MF, Ghule PN, Lee K, Vacek P, Sprague BL, et al. (2023) Identification of molecularly unique tumor-associated mesenchymal stromal cells in breast cancer patients. PLoS ONE 18(3): e0282473. https://doi.org/10.1371/journal.pone.0282473
Editor: Gianpaolo Papaccio, Università degli Studi della Campania, ITALY
Received: December 8, 2022; Accepted: February 16, 2023; Published: March 20, 2023
Copyright: © 2023 Gordon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The raw RNA sequencing data have been deposited in dbGAP phs002225 or GSE183931 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE183931).
Funding: This work was funded by National Cancer Institute/NCI/NIH grant 1 U01 CA196383-01 (J.L.S/D.L.W/B.L.S), P01 CA240685 (G.S.S./J.L.S) and American Cancer Society Institutional Research Grant (IRG-14-196-01) (J.A.R.G). The Vermont Integrative Genomics Resource Massively Parallel Sequencing Facility was supported by the University of Vermont Cancer Center, Lake Champlain Cancer Research Organization, UVM College of Agriculture and Life Sciences, and the UVM Larner College of Medicine. The Harry Hood Bassett Flow Cytometry and Cell Sorting (FCCS) Facility at UVM Larner College of Medicine was supported by National Institute of Health Shared Instrumentation Grants, NIH S10-ODO026843 and NIH S10-ODO02976. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
