Additional file 2 of Simultaneous targeting of HER family pro-survival signaling with Pan-HER antibody mixture is highly effective in TNBC: a preclinical trial with PDXs
Creators: name: Reddy, Tejaswini P.
name: Choi, Dong S.
name: Anselme, Ann C.
name: Qian, Wei
name: Chen, Wen
name: Lantto, Johan
name: Horak, Ivan D.
name: Kragh, Michael
name: Chang, Jenny C.
name: Rosato, Roberto R.
Publisher: name: figshare
Year: 2020
Subjects: subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Computational Biology

description: Additional file 2 Figure 1. A, Microarray analysis of RNA gene expression corresponding to PDX samples before treatment. Note the differences in expression of PDXs having shown tumor regression (i.e., BCM-3936, BCM-4913 and MC1; Subgroup 1) vs. the rest of the PDXs (Subgroup 2); PDX BCM-4195 does not express EGFR, HER2, or HER3 and was added to the analysis for comparison only. B, gene expression analysis by Ingenuity Pathway Analysis (Qiagen) comparing BCM-3936, BCM-4913 and MC1 (subgroup 1) vs. the remaining PDXs; it shows among the top molecules a marked reduction of genes related to the AKT/PKB survival pathway including the PTEN pathway, and increased expression of PTK2 (FAK). Supplemental Figures 2-16. Time course analysis of the therapeutic response corresponding to each of the 15 TNBC PDXs used in the present study. A, graph displaying the time-course analysis of tumor growth; B, Western blot analysis of HER family members and associated signaling events; and C, IHC of EGFR and HER3 proteins. Low passage TNBC PDX tumor samples (2 mm × 2 mm) were transferred into the right mammary fat pad of mice for engraftment. Once tumors reached an average size of 150-200 mm3, mice were randomized (n ≥ 3 per group) and treated following the three, one-week cycles design, consisting of 3 times/week IP injection of either formulation buffer (Vehicle control) or Pan-HER (50 mg/kg). Mouse weight was recorded and tumor volumes were measured and calculated as described in Materials &amp; Methods twice weekly. Tumor volume fold change was calculated based on the baseline tumor volumes for each arm. Two-way ANOVA was used for a statistical analysis. At the end of the 3-cycle treatment, the animals were sacrificed and tumors collected for further Western blot and IHC analyses. Supplemental Figures 17-20. EGFR (A) and NF-κB (B) pathway-focused RT-PCR gene expression analysis of representative TNBC PDXs RNA samples collected before and after Pan-HER treatment. RNA samples corresponding to representative PDX tumor model BCM-2147 and BCM-2665 (Subgroup 2), and BCM-3555 and BCM-4913 (Subgroup 1) were extracted from 3 independent mice( PDX)/group treated with either Vehicle control or Pan-HER for 3 cycles at the end of the experiment (day 21 after the initial injection). Triplicate RT-PCR plates were run and relative fold changes of Pan-HER- vs. Vehicle control-treated samples for each gene were analyzed by Ingenuity Pathway Analysis (IPA; Qiagen). Genes shown in green represent those significantly down-regulated, while those in red up-regulated. A 2-fold change cut-off in gene expression threshold was considered as significantly changed (p &lt; 0.001). Further details, as well as a similar analysis performed in 3 additional PDX models are shown as Supplemental Figures.