Id1 Marks Luminally Incorporated, Mature EPCs in the Nascent Tumor Vasculature. A Upper, Microscopy showing vasculature and BM-derived Id1^+/GFP+ CD31^low CD11b⁻ BM-EPCs as part of the tumor-stroma (arrows) of LLC tumors (day 15) implanted into Id1^+/GFP+ BMT mice (n=10). Scale bar, 50µm. Lower, Microscopy of a tumor vessel showing an incorporated BM-derived mature EPC (Id1^+/GFP+ CD31⁺ CD11b⁻, arrow) in LLC tumors (day 15) implanted into Id1^+/GFP+ BMT mice. Scale bar, 20µm. B, High resolution (63×) transverse immunofluorescent image of tumor vessel showing an incorporated BM-derived mature EPC (Id1^+/GFP+ CD31⁺ CD11b⁻, arrow) in LLC tumors (day 15) implanted into Id1^+/GFP+ BMT mice. Scale bar, 20µm. C, Summary of FACS analysis showing contribution of Id1⁺ mature EPCs in functional vessels, by Isolectin staining in LLC tumor (day 6) in Id1pr/p-GFP BMT mice. D, Representative scatter plots showing derivation of GFP gates. SSCA, side scatter values.

Id1pr/p shRNA-Mediated Suppression of VEGFR2 and Id1 in the EPCs Results in Angiogenesis Inhibition and Impaired Tumor Growth. A Upper, Schematic of Id1pr/p-GFPΩ LV vector with Id1 promoter driving GFP and miR30-based Ω expression. Lower, LLC tumor growth in Id1pr/p-GFPNSΩ, Id1pr/p-GFPId1Ω and Id1pr/p-GFPVEGFR2Ω BMT mice. Data represented as mean volume±S.E.M (P=0.0117 by Student’s t-Test, n=5 per group). B, LLC tumor morphology in Id1pr/p-GFPNSΩ, Id1pr/p-GFPId1Ω and Id1pr/p-GFPVEGFR2Ω BMT mice, Scale bar, 5mm. C, FACS analysis of PB showing number of CEPs (c-kit⁺ VEGFR2⁺), and HPCs (c-kit⁺CD45⁺) from LLC tumor challenged Id1pr/p-GFPΩ mice. Data is represented as mean number of cells per 1×10⁵±S.E.M. (n=5 per group; P values, by Student’s t-Test). This experiment was repeated, similar trends observed. D, CD31⁺ immunostaining showing a higher vessel density in Id1pr/p-GFPNSΩ LLC tumors in comparison to Id1pr/p-GFPId1Ω and Id1pr/p-GFPVEGFR2Ω LLC tumors (n=15), Scale bar, 100µm. Data represented as average number of vessels per field±S.E.M. and analyzed by Students t-test.

Id1 Marks EPCs in the PB, BM and Tumor-Stroma. A Upper, Schematic of Id1pr/p-GFP LV vector: Id1pr/p driving GFP expression. cPPT, central polypurine tract; WPRE, woodchuck hepatitis virus post transcription regulatory element; LTR, long terminal repeat; SIN, self inactivating. Lower, Microscopy showing recruitment of Id1pr/p-GFP⁺ cells to early nonvascularized LLC tumor periphery (arrows, day 8, n=15) in Id1pr/p-GFP BMT mice. Scale bar, 50µm. B Upper, Schematic of Id1pr/p-RFP LV vector: Id1pr/p driving monomeric RFP/ mCherry™ expression. Lower, Microscopy showing recruitment of Id1pr/p-RFP⁺ cells to early nonvascularized LLC tumor periphery (arrows, day 8, n=15) in Id1pr/p-RFP/ACTb-EGFP BMT mice. Scale bar, 50µm. High resolution (63×) image showing the Id1pr/p-RFP⁺ can be used to mark and distinguish BM-derived ACTb-EGFP⁺ VE-Cadherin⁺ BM-EPCs (arrow) from other BM cells in within the tumor-stroma. Scale bar, 10µm. C top, Image showing that the LV-Id1pr/p-GFP construct can be used to mark Id1 protein expressing VE-cadherin⁺ BM-EPCs (arrow) in the tumor stroma. Bar, 10 µm. Bottom, high-resolution (×63) microscopic images of cytospun BM and PB from LLC tumor�challenged ACTb-EGFP/Id1pr/p-RFP BMT animals, showing that Id1pr/p⁺ EPCs in the BM and PB express nuclear Id1 protein (white arrows). Bar, 20 µm. D Upper, Microscopy showing vasculature and BM-derived Id1^+/GFP+ VE-Cadherin⁺ CD31^low BM-EPCs as part of the tumor-stroma (arrows) of LLC tumors (day 15) implanted into Id1^+/GFP+ BMT mice (n=10). Scale bar, 50µm. Lower, High resolution (63×) of BM-derived Id1^+/GFP+ VE-Cadherin⁺ CD31^low BM-EPC (arrow) in the tumor-stroma of LLC tumors (day 15) implanted into Id1^+/GFP+ BMT mice. Scale bar, 10µm.

Selective Elimination of Id1pr/p⁺ EPCs by Delivery of a Suicide Gene HSV-tk Impairs Tumor Growth. A Upper, Schematic of Id1pr/p LV vector, with Id1pr/p driving GFP and HSV-tk expression. Lower, LLC tumor growth (Mean±S.E.M) and morphology in Id1pr/p-GFPITK BMT mice (n=10) and Id1pr/p-GFP BMT mice (n=5), either treated with GCV (+GCV) or untreated (−GCV). Similar trends observed in repeat experiment. B, FACS analysis of PB from tumor challenged Id1pr/p-GFPITK (+GCV), Id1pr/p-GFPITK (−GCV), and GCV control Id1pr/p-GFP (+GCV) BMT mice, showing number of mobilized CEPs (c-kit⁺ VEGFR2⁺) and HPCs (c-kit⁺ CD45⁺). A total of 1×10⁶ cells were analyzed per animal. Data is represented as mean number of cells per 1×10⁵ PBMNCs±S.E.M (n=5 per group); analyzed by Students t-test. C Upper, Microscopic analysis of EPCs (VE-Cadherin⁺ GFP⁺) from the BM following GCV treatment in Id1pr/p-GFPITK BMT mice. Lower, FACS analysis of the BM from tumor challenged Id1pr/p-GFPITK (− or + GCV) BMT mice, showing the number of EPCs (c-kit⁺ VEGFR2⁺), myeloid cells (CD11b⁺). Data showing significant difference between GCV treated Id1pr/p-GFPITK and untreated animals (P=0.040, by Students t-Test). Numbers are normalized per 1×10⁶ BM mononuclear cells. D, CD31⁺ immunostaining showing a lower vessel density in Id1pr/p-GFPITK +GCV LLC tumors in comparison to Id1pr/p-GFPITK −GCV and Id1pr/p LLC tumors (n=15), Scale bar, 100µm. Data represented as average number of vessels per field±S.E.M. and analyzed by Students t-test.