Immature myeloid cells derived from mouse placentas and malignant tumors demonstrate similar proangiogenic transcriptional signatures  Ofer Fainaru, M.D., Ph.D., Niv Pencovich, Ph.D., Shay Hantisteanu, M.Sc., Golan Yona, Ph.D., Mordechai Hallak, M.D.  Fertility and Sterility  Volume 99, Issue 3, Pages 910-917.e2 (March 2013) DOI: 10.1016/j.fertnstert.2012.11.018 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Isolation of immature myeloid cells from placenta and tumor tissues. (A) Placentas from day 14 pregnancies (n = 3) and (B) subcutaneous Lewis lung carcinoma tumors (∼ 100 mm3) (n = 3) were harvested and enzymatically digested. Single cell suspension CD45+ hematopoietic cells were gated (upper), and of CD11b+Gr1+ immature myeloid cells (lower) were isolated using a flow cytometry-based cell sorter. Fertility and Sterility 2013 99, 910-917.e2DOI: (10.1016/j.fertnstert.2012.11.018) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Immature myeloid cell (IMC) global gene expression. (A) Principal component analysis of gene expression sets derived from placenta IMCs (n = 3), tumor IMCs (n = 3), and primary muscle cells (n = 4). Note the proximity of muscle and placenta IMC gene sets indicating molecular similarity. (B) Analysis of similarity using the mass–distance algorithm resulted in three major gene sets: (a) genes that are similarly expressed in placenta IMCs and tumor IMCs but are not expressed in primary muscle cells; (b) genes that are expressed in tumor IMCs but not in placenta IMCs; and (c) genes that are expressed in placenta IMCs but not in tumor IMCs. (C) Cluster analysis of gene sets (a, b, c) using CLICK algorithm, resulted in four gene clusters. Biological repeats of placenta IMCs (P), tumor IMCs (T), primary muscle cells (M) are presented. Fertility and Sterility 2013 99, 910-917.e2DOI: (10.1016/j.fertnstert.2012.11.018) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Unique gene expression sets of immature myeloid cells specific to hosting tissue. Heatmaps demonstrating genes selectively expressed in IMCs derived from tumors but not from placentas (clusters 2 and 4) (A); and genes selectively expressed in IMCs derived from placentas but not from tumors (cluster 3) (B). Note that genes from these clusters were specific for IMCs and were not expressed by primary muscle cells. Fertility and Sterility 2013 99, 910-917.e2DOI: (10.1016/j.fertnstert.2012.11.018) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Placenta and tumor immature myeloid cell highly expressed genes. (A) Genes that are highly expressed in placenta and tumor IMCs but not in muscle cells are represented in cluster 1. Gene Ontology (GO) functional annotations for this cluster are presented; highly enriched and relevant functional annotations are indicated. (B) Quantitative polymerase chain reaction (PCR) of expression in placenta and tumor IMCs relative to muscle cells of important selected genes from cluster 1. Data represent means ± SD of two biological repeats performed in triplicates. (C) Quantitative PCR of expression in placenta- versus tumor-derived IMCs of key genes from sets b and c (Fig. 2B). Data represent means ± SD of three biological repeats performed in triplicates. *P<.05. Fertility and Sterility 2013 99, 910-917.e2DOI: (10.1016/j.fertnstert.2012.11.018) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Assesment of similarity using the mass–distance algorithm. (A) The distribution of gene expression values in tumor immature myeloid cells (IMCs), placenta IMCs, and primary muscle cells. The distributions reflect the presence of two populations. The main one consists of inactive genes, or genes whose expression is normal, and gives rise to the background distribution on the left. The second peak corresponds to the population of active, overexpressed genes. The threshold between the two populations is set to 5.8. (B) The mass–distance is a measure of probability, which defines the difference between two measurements as the probability to observe by chance a measurement between them. It calibrates the nominal difference between measurements based on the total distribution of samples, thus adjusting to the specific characteristics of the data. As such, it is more sensitive in discerning true similarities from chance similarities compared to other common measures of distance or similarity. For example, in the dataset plotted above the two measurements p1 and p2 are statistically more similar to each other than the two measurements p3 and p4 although p2 - p1 = p4 - p3. That is because there are fewer measurements between p1 and p2, and therefore fewer instances that have similar properties. The probability mass (shaded area) in each case is given by the integral over the background distribution. (C) For genes to be includes in set b, the mass–distance between the minimal expression value over all replicas in tumor IMCs and the maximal value over all replicas in placenta IMCs has to be at least 0.1 (equal to 10% of the total distribution). Fertility and Sterility 2013 99, 910-917.e2DOI: (10.1016/j.fertnstert.2012.11.018) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Angiogenesis-related genes in sets (a), (b), and (c). A list of selected, angiogenesis-related genes that are highly expressed in placenta- and tumor-derived immature myeloid cells (IMCs), only in tumor-derived IMCs, and only in placenta-derived IMCs. Normalized means of raw expression data is presented for each tissue. Dark gray represent high expression values relative to the relevant tissue. Fertility and Sterility 2013 99, 910-917.e2DOI: (10.1016/j.fertnstert.2012.11.018) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions