Studies with transplanted mouse bone marrow stem cells. (A) Scheme of experiment. Whole bone marrow cells (B-G) or Lineage-depleted (Lin−) cells (G,H) of male neu-tg mice were transplanted into lethally irradiated female neu-tg mice by tail injection. Six weeks after HSC engraftment, MMC tumors were established by injection of 5 × 105 MMCs. (B) Three weeks after tumor transplantation, tumor sections were analyzed for mouse Y chromosome (left), mouse CD45, and laminin (middle). (B right) Y chromosome signals are shown in a black and white exposure to better visualize their relation to tumor nests. (C) Tumor localization of transplanted mouse bone marrow cells that were transduced with a VSV-G pseudotyped b-Gal–expressing lentivirus vector (LV-bGal). Tumor sections were analyzed for bGal by X-Gal staining (left) and immunofluorescence with anti-bGal and anti-CD45 antibodies (middle) or anti-bGal and antilaminin antibodies (right). The scale bar represents 100 μm. (D) Mouse bone marrow cells were transduced ex vivo with the relaxin-expressing lentivirus vectors (LV-Rlx) or LV-bGal and transplanted into irradiated neu-tg mice. Six weeks later, MMCs were injected subcutaneously. Two days after MMC transplantation mice received Dox, and tumor size was measured (n = 7 per group). (E,F) Three weeks after tumor cell transplantation, morphometric analysis of collagen IV was performed on tumor sections (E), and the number of CD45+ TILs inside tumor nests in direct contact with tumor cells was counted on 10 sections stained for CD45 and laminin (n = 7 per group; F). (G) Flow cytometric analysis for the T-cell marker CD3 and the HSC marker CD117 (cKit) in whole bone marrow and Lin− cells obtained after 1 (1x) or 2 rounds (2x) of magnetic cell sorting using a Lineage-cell depletion kit. Bone marrow was harvested from femurs of neu-tg mice 2 days after 5-FU treatment (n = 3 per group). (H) LV-bGal- or LV-Rlx-transduced Lin− cells were transplanted into lethally irradiated neu-tg mice. Six weeks later, MMC tumors were established by mammary fat pad injection of 5 × 105 MMCs in Matrigel. Both experimental groups received Dox treatment. Shown is the tumor volume (n = 5 per group). Error bars represent SD.
