Supplemental Figure S3: IPA pathway highlights genes relevant to, Cell Migration, Angiogenesis and Morbidity or mortality for Treatment groups at Week 2 compared to Untreated Week 2. Significantly altered genes ([2-fold change, p < 0.01) are shown. White indicates no significant changes, Red indicates upregulation, Blue indicates downregulation. Black arrows indicate genes of interest. More genes exist in these pathways than are displayed, for ease of visualization only genes which showed significant changes in expression in at least 2 treatment groups are presented. Genes may be relevant to more than one pathway (PLAUR) is relevant to Organismal Death and Vasculogenesis, but to avoid redundancy is shown once.

Supplemental Figure S6: Graphs of proteins and phosphoproteins that previously showed impact on cancer proliferation or death. Trends towards significance only. Normalized to intensity. For JUN, microarray data showed that Drug Week 2 and Drug+Rad Week 1 were significantly decreased ([FC} > 2, p < 0.05) compared to respective Untreated tumors.

Supplemental Figure S4: Heat Shock protein gene expression was significantly modified by treatment over time. Statistical difference (2-fold change, p < 0.01) indicated with (*). No radiation samples showed statistically significant changes compared to time-matched Untreated tumors, though there was a trend towards significant downregulation for HSPB11 and HSP90AA1 in Rad Week 2 samples compared to UT_Wk2. HSPA1A was significantly upregulated in Drug+Rad Week 1 and Week 2 samples compared to respective UT_Wk1 and 2 samples. HSPA1B was also upregulated in Drug and Drug+Rad at Week 1 and Week 2 compared to respective UT_Wk1 and UT_Wk2. HSP90AA1 was only significantly downregulated at Drug Week 2. For HSPB11 only Drug+Rad Week 1 and Week 2 were significantly downregulated. HSPA14 showed significant downregulation at Drug and Drug+Rad Week 1 and Drug Week 2 compared to respective controls.

Supplementary Table of phosphoproteomic data

Supplemental Figure S1: IPA pathway highlights genes relevant to Organismal Death, Viral infection and Vasculogenesis for Untreated groups at Week 1 and Week 2 compared to Control Day 1. Significantly altered genes (2-fold change, p < 0.01) are shown. White indicates no significant change, Red indicates upregulation, Blue indicates downregulation. Black arrows indicate genes of interest.

Supplemental Figure S2: IPA pathway highlights genes relevant to Organismal Death, Viral Infection and Vasculogenesis for Treatment groups at Week 1 compared to Control Day 1. Significantly altered genes (2-fold change, p < 0.01) are shown. White indicates no significant change, Red indicates upregulation, Blue indicates downregulation. Black arrows indicate genes of interest. Drug+Rad and Drug alone caused significantly more gene up/down regulation than Rad alone. More genes exist in these pathways than are displayed, for ease of visualization only genes which showed significant changes in expression in at least 2 treatment groups are presented. Genes may be relevant to more than one pathway (PLAUR) is relevant to Organismal Death and Vasculogenesis, but to avoid redundancy is shown once.

Supplemental Figure S5. Proteomic and Phospho-proteomic comparison of changes observed comparing UT_Wk1 to UT_Wk2 (A), Treated Week 1 to UT_Wk1 (B) and Treated Week 2 to UT_Wk2 (C). Trends towards significance only. Normalized to intensity. No notable change is shown in white, upregulation is red, downregulation is blue. Supplemental Figure 4A indicates change in UT_Wk2 compared to UT_Wk1, interestingly p53 and PDL-1 protein expression decrease over three days. Supplemental Figure 6A indicates change in Untreated Week 2 compared to Untreated Week 1, interestingly p53 and PDL-1 protein expression show a decreasing trend.

Abstract Treatments involving radiation and chemotherapy alone or in combination have improved patient survival and quality of life. However, cancers frequently evade these therapies due to adaptation and tumor evolution. Given the complexity of predicting response based solely on the initial genetic profile of a patient, a predetermined treatment course may miss critical adaptation that can cause resistance or induce new targets for drug and immunotherapy. To address the timescale for these evasive mechanisms, using a mouse xenograft tumor model, we investigated the rapidity of gene expression (mRNA), molecular pathway, and phosphoproteome changes after radiation, an HSP90 inhibitor, or combination. Animals received radiation, drug, or combination treatment for 1 or 2 weeks and were then euthanized along with a time-matched untreated group for comparison. Changes in gene expression occur as early as 1 week after treatment initiation. Apoptosis and cell death pathways were activated in irradiated tumor samples. For the HSP90 inhibitor and combination treatment at weeks 1 and 2 compared with Control Day 1, gene-expression changes induced inhibition of pathways including invasion of cells, vasculogenesis, and viral infection among others. The combination group included both drug-alone and radiation-alone changes. Our data demonstrate the rapidity of gene expression and functional pathway changes in the evolving tumor as it responds to treatment. Discovering these phenotypic adaptations may help elucidate the challenges in using sustained treatment regimens and could also define evolving targets for therapeutic efficacy.

Subcellular localization of AKT and mTOR after radiation. A, Western blots of mTOR, phospho-AKT S473 and AKT 2 h after 3 fractions of 2 Gy in 3D-grown PC3 cells after CRISPR/Cas9-mediated depletion of mTOR (crMTOR) using different targeting sequences. A non-targeting sequence (NTS) was used as control. β-actin expression was evaluated to ensure equal sample loading. B, Colony formation data of 3D colony formation assays. Cells were irradiated either with 6 Gy single dose or with 3 fractions of 2 Gy (2 Gy per day). Results show mean {plus minus} SD (n = 3, ** P < 0.01, Student''s t test). (See also Figure 6)

Oligonucleotides used for CRISPR/Cas9 gene editing

Subcellular localization of AKT and mTOR after radiation. Immunofluorescence staining of AKT and mTOR in irradiated and unirradiated DU145 and PC3 cells. Nuclear staining was performed with DAPI.

PDF file - 621K, Supplementary Figure S1. Treatment with Cetuximab inhibits EGF-mediated EGFR phosphorylation under 2D and 3D cell culture conditions in a cell line-dependent manner. Supplementary Figure S2. Induction of Cetuximab-mediated apoptosis in non-irradiated and irradiated 3D grown SCC cells. Supplementary Figure S3. EGFR inhibition with Cetuximab results in minor Caspase 3 cleavage. Supplementary Figure S4. Treatment with Cetuximab modulates tyrosine and serine protein phosphorylation. Supplementary Figure S5. JNK inhibition using siRNA or pharmacological inhibitor (SP600125) reduces cell survival of SCC cells. Supplementary Figure S6. JNK inhibition impairs Cetuximab-mediated c-Jun S63 phosphorylation. Supplementary Figure S7. JNK2 knockdown sensitizes UTSCC5 cells to Cetuximab-mediated radiosensitization. Supplementary Figure S8. EGFR and JIP-4 co-precipitate in untreated FaDu 3D cell cultures

S1. Growth curves of PC3 xenograft tumors after radiation. S2. AIIB2 binds to prostate cancer cells. S3. Integrin expression is elevated after radiation in vitro and in vivo. S4. Cell cycle distribution is unchanged in PC3 long-term survivors (in vitro).

PDF file, 606K, Methods, Figures, Figure legends, Tables Fig. S1. A, EGFR protein expression in A549 and H1299 cells evaluated by Western blot. Fig. S2. A, A549 cells were cultured in the presence (+FCS) or absence (-FCS) of FCS and B, additionally treated with Cetuximab (5 microg/ml) for 24 h or 48 h. Fig. S3. Confocal fluorescence images of EGFR localization Fig. S4. Western blot analysis of A549 cells treated with Cetuximab Fig. S5. Immunofluorescence staining of CREB and phosphorylated CREB in A549 cells 48 h after treatment with Cetuximab Fig. S6. Promotor activity Table S1. List of Data sets Table S2. List of specific primers and probes.

Supplementary Methods, Figures 1-15, Tables 1-9 from Three-Dimensional Cell Growth Confers Radioresistance by Chromatin Density Modification

Radiation schedule for the cell culture and xenograft experiments. Human prostate cancer cells were either plated or injected subcutaneously into the flanks of the right hind legs of athymic nude mice. After 24 h (in vitro) or tumors reached a tumor size of 5 mm x 5 mm (in vivo), tumors were irradiated with different fractionation regimens including a single dose of 10 Gy at day 5, 10 fractions of 1 Gy (2 fractions per day), or 5 fractions of 2 Gy (1 fraction per day). Unirradiated cells or tumors were used as control.

Metabolic data from PC3 cells 6h, 24h, and 48h after SD or MF radiation. Samples were sent to Metabolon for processing. Results indicate decrease in glycolysis intermediates in SD sample at 6h. No significant changes to TCA cycle intermediates. No significant changes to ATP at any time points measured.

PurposePrevious research has highlighted the impact of radiation damage, with cancer patients developing acute disorders including radiation induced pneumonitis or chronic disorders including pulmonary fibrosis months after radiation therapy ends. We sought to discover biomarkers that predict these injuries and develop treatments that mitigate this damage and improve quality of life.Materials and methodsSix- to eight-week-old female C57BL/6 mice received 1, 2, 4, 8, 12 Gy or sham whole body irradiation. Animals were euthanized 48 h post exposure and lungs removed, snap frozen and underwent RNA isolation. Microarray analysis was performed to determine dysregulation of messenger RNA (mRNA), microRNA (miRNA), and long non-coding RNA (lncRNA) after radiation injury.ResultsWe observed sustained dysregulation of specific RNA markers including: mRNAs, lncRNAs, and miRNAs across all doses. We also identified significantly upregulated genes that can indicate high dose exposure, including Cpt1c, Pdk4, Gdf15, and Eda2r, which are markers of senescence and fibrosis. Only three miRNAs were significantly dysregulated across all radiation doses: miRNA-142-3p and miRNA-142-5p were downregulated and miRNA-34a-5p was upregulated. IPA analysis predicted inhibition of several molecular pathways with increasing doses of radiation, including: T cell development, Quantity of leukocytes, Quantity of lymphocytes, and Cell viability.ConclusionsThese RNA biomarkers might be highly relevant in the development of treatments and in predicting normal tissue injury in patients undergoing radiation treatment. We are conducting further experiments in our laboratory, which includes a human lung-on-a-chip model, to develop a decision tree model using RNA biomarkers.

We present the case of a woman with metastatic adenoid cystic carcinoma who received stereotactic ablative radiation therapy with a total dose of 50 Gy in 4 fractions to 2 lung metastases and developed symptomatic left phrenic nerve injury 2 years after radiation. The maximum dose to the approximate location of the phrenic nerve was 57.7 Gy, which corresponds to a biologically effective dose for late effects (using α/β ratio = 3) of 335.14 Gy. Here, we discuss the case, planning considerations by radiation oncologists and medical physicists, and the multidisciplinary medical management of this patient.

PDF file - 109K, Supplementary Table S1. Plating efficiencies of SCC cells differ between 2D and 3D cell culture conditions cell line-dependently. Supplementary Table S2. JIP-4, Grb2 and STE20-like kinase bind to EGFR in untreated FaDu 3D cell cultures as determined by immunoprecipitation and subsequent mass spectrometry. Supplementary Table S3. Knockdown of JIP-4 and JNK2 in combination with Cetuximab synergistically impact on radiation survival of SCC cells