Supplemental Methods Immunohistochemical Analyses Immunostaining was performed on tumor biopsy samples arranged in a tissue-microarray format or on prostatectomy sections obtained post-study. Briefly, formalin-fixed, paraffin-embedded slides were rehydrated, blocked first with 3% H 2 2, followed by % goat serum. Slides were then incubated with a primary antibody against insulin receptor (1:1, #sc-7342, Santa Cruz Biotechnology) for 16h at 4 C, and followed by corresponding secondary antibody and a Universal Elite avidin-biotin complex kit (Vector Laboratories) according to manufacturer s protocol. The reaction was visualized with DAB, counterstained with hematoxylin, dehydrated and mounted with Permount (Fisher Scientific). Following staining, slides were scanned using the APERIO system (APERIO, Vista CA). Analysis of staining was performed as previously described (Zhang X, et al, 211 PLoS One 6:e2797)
Supplemental Figure Legends: Figure 1. Insulin receptor immunohistochemistry, A) antibody negative control in human prostate, B) antibody positive control, human liver, C) prostate tissue from TAPS patient, D) prostate tissue from cixutumumab treated patient. Note no significant differences between TAPS tissue and cixutumumab tissue were detected. Figure 2. Decreases in PSA were similar between the Neo ADT cohort and the Neo ADT + cixutumumab (A12) cohort (A & B). No significant differences were detected among the different BMI categories in the A12 cohort (C & D), although pre-adt PSA levels trended toward significant in the obese BMI group (ANOVA, p =.9; t-test, p =.6 for obese vs overweight). Figure 3. Decreases in serum testosterone were similar between the Neo ADT cohort and the Neo ADT+A12 cohort (A & B). Likewise no significant differences were seen among BMI categories in the Neo ADT+A12 cohort (C & D). Figure 4. Effects of IGF-1R blockade and hormone therapy on IGF pathway homeostasis in the different BMI categories. Serum levels of IGF-1, -II, and IGFBP-3 were measured by ELISA. No significant differences were detected among the BMI categories for these serum factors. Figure. Body mass index affects serum blood glucose levels in response to cixutumumab treatment. Serum levels of blood glucose were determined by ELISA. p <., Neo ADT cohort as a whole compared to Neo ADT+A12 obese BMI patients. # = trending toward significance (p =.23).
Figure 6. Body mass index affects serum insulin levels in response to cixutumumab treatment. Serum levels of insulin were determined by ELISA. p <., p <.1, Neo ADT cohort as a whole compared to Neo ADT+A12 patients by BMI. Figure 7. Body mass index affects serum C-peptide levels in response to cixutumumab treatment. Serum levels of C-peptide were determined by ELISA. p <., p <.1, Neo ADT cohort as a whole compared to Neo ADT+A12 patients by BMI.
Supplemental Figure 1. A B C D
Supplemental Figure 2. A. B. PSA 3 2 2 1 1 Neo ADT Neo ADT + A12.4.3.3.2.2.1.1. PSA at time of Surgery Neo ADT Neo ADT + A12 C. D. 6 4 3 2 1 PSA in A12 cohort Normal Overweight Obese.6..4.3.2.1 PSA at time of Surgery
Supplemental Figure 3. A. Testosterone B. 4 3 Neo ADT 2 Neo ADT + A12 1-1.3.3.2.2.1.1. Testosterone at time of Surgery Neo ADT Neo ADT + A12 C. Testosterone in A12 cohort D. 6 4 Normal 3 Overweight 2 Obese 1-1.3.2.2.1.1. Testosterone at time of Surgery
Supplemental Figure 4. 1 IGF-I 8 6 4 2 Normal Overweight Obese 12 1 8 6 4 2 IGF-II Normal Overweight Obese 6 4 3 2 1 IGFBP-3 Normal Overweight Obese
Supplemental Figure. 16 14 12 1 8 6 Blood Glucose in Normal BMI Patients normal Blood Glucose in Overweight BMI Patients 16 1 14 13 12 11 1 9 8 7 6 overweight 16 1 14 13 12 11 1 9 8 7 6 Blood Glucose in Obese BMI Patients # obese
Supplemental Figure 6. 8 7 6 4 3 2 1 Insulin in Normal BMI Patients normal 8 7 6 4 3 2 1 Insulin in Overweight BMI Patients overweight 8 7 6 4 3 2 1 Insulin in Obese BMI Patients obese
Supplemental Figure 7. 1 9 8 7 6 4 3 2 1 C-peptide in Normal BMI Patients normal 1 9 8 7 6 4 3 2 1 C-peptide in Overweight BMI Patients overweight 1 9 8 7 6 4 3 2 1 C-peptide in Obese BMI Patients obese