The Hematopoietic Origin of Tumor Fibroblasts and Pericytes.
Authors: LaRue Amanda C. , Masuya Masahiro , Fleming Paul A. , Visconti Richard P. , Ogawa Makio , Drake Christopher J. 
Published: October 2019

Abstract
The role of the tumor stroma in solid tumor progression and invasion has become an important area of current research and has potential as a therapeutic target. The tumor stroma is composed of a variety of cell types including immune cells, inflammatory cells, fibroblasts and vascular cells (i.e., endothelial cells and pericytes). It was recently reported that crude bone marrow cells have the ability to transdifferentiate into fibroblast-like cells within the tumor capsule and fibroblast-like pericytes associated with the tumor vasculature. However, it is unclear whether these cells are of hematopoietic stem cell (HSC) or stromal/mesenchymal stem cell (SSC) origin. We tested the hypothesis that tumor fibroblasts are derived from HSCs by combining a single HSC transplantation strategy with murine tumor models. For these studies, single cells were sorted from transgenic enhanced green fluorescent protein (EGFP) murine bone marrow based on surface antigen expression (Lin−c-kit+Sca-1+CD34−) or Hoechst 33342 dye exclusion (side population). Individual cells were deposited into wells of a 96-well plate and cultured for one week. Each viable clone (derived from a single stem cell) was then transplanted into a lethally irradiated mouse. Two months later, mice showing high levels (60–90%) of multilineage hematopoietic reconstitution were injected subcutaneously with either murine melanoma or Lewis lung carcinoma cells. Murine melanoma cells (K1735-M2) were a generous gift from Mary Hendrix (University of Iowa, IA) and murine Lewis lung carcinoma (LLC1) were obtained from ATCC (Manassas, VA). After 7–10 days, tumors were extracted and processed for paraffin sectioning. Analysis of thin 3-micrometer sections using differential interference contrast (DIC) and epifluorescence microscopy showed two major populations of EGFP+ cells within the tumor. The first was found within the tumor capsule and had morphological characteristics of fibroblasts. To determine if these cells were indeed fibroblasts, in situ hybridization studies were conducted using a riboprobe for the alpha 1 subunit of type I collagen. Type I collagen mRNA expression was detected in a subpopulation of EGFP+ cells in the tumor capsule, indicating that these HSC-derived EGFP+ cells are fibroblasts. The second cell type was found associated with the blood vessels of the tumor. Analysis of the morphology of these cells using high magnification DIC and epifluorescence microscopy suggested that these cells were not endothelial cells, but support cells (pericytes) in close contact with the endothelium. Immunohistochemical examination of these cells using antibodies to CD31, a protein expressed by endothelial cells, and laser scanning confocal microscopy confirmed that these cells were not endothelial cells (EGFP+/CD31−). Transplantation of 100 Lin−c-kit+ Sca-1+CD34− bone marrow cells without culture produced the same results. These findings establish a hematopoietic origin of fibroblasts comprising the tumor capsule and pericytes of the tumor vasculature.