The entire study was performed in compliance with Institutional and Federal guidelines for clinical research. All patient tissue and blood samples were drawn with informed consent based on a local IRB approved consent form. .
SERUM SAMPLE COLLECTION
Serum samples were obtained from 8 patients, all having KRAS-mutated mCRC. Five (5) patients had received reovirus as part of a phase 1 clinical trial (NCT01274624; 11). Three (3) patients were not enrolled in the trial but did receive equivalent background chemotherapy (i.e., FOLFIRI and bevacizumab).
Reovirus was supplied by Oncolytics Biotech, Inc. as a translucent to clear, colorless to light blue liquid in vials containing 7.2 X 1011 tissue culture infective dose (TCID50) per ml of reovirus in a phosphate- buffered solution and stored at minus 70oC. Reovirus was administered as a 60-minute infusion for 5 consecutive days every 28 days, at a tissue culture infective dose (TCID50) of 3x1010/day. Plasma was collected pre-reovirus, at 24 and 48 hours, and 7 and 14 days after first dose of reoviru
To assess the immune-modulating effects of reovirus, blood was drawn into cell preparation tubes (CPT) (BD Vacutainer® CPT™, Mononuclear Cell Preparation Tubes, (manufacturer # 362753) to isolate peripheral blood mononuclear cells (PBMC). Fluorescence activated cell sorting (FACS) assay was performed using fluorophore labeled antibody staining for T helper lymphocyte (FITC-CD4; catalog # 11-0049; Thermofisher-eBiosciences), cytotoxic T lymphocyte (PE-CD8; catalog # 12-0088; Thermofisher-eBiosciences), activated cytotoxic T lymphocyte (CD70-eFluor 660; catalog # 50-0709; Thermofisher-eBiosciences), mature dendritic cell (CD123-PE-Cy7; catalog # 25-1239; Thermofisher- eBiosciences) and Natural killer (NK) cells (CD56-eFluor 450 catalog # 48-0566; Thermofisher- eBiosciences) along with live dead marker (FVD-eFluor 780; catalog # 65-0865 Thermofisher- eBiosciences). The staining and data acquisition was performed within 3 hours of sample collection. Flo Jo software (version 9.8.1) was used for all analysis and gating was maintained unaltered throughout the entire analysis.
Cytokine assay – serum samples (post PBMC isolation) from the 5 patients who received reovirus were centrifuged for 30 minutes at 12,500 G. Next, 50 mL of supernatant was added to a 96-well plate (samples were added in triplicate). EMD Millipore beads coated with the following cytokines were then added to each sample (Cat#HCYTOMAG-60K-25): IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, GM-CSF, IFN-alpha 2, IFN gamma,
TNF alpha, MCP-1, MCP-3, MIP-1 alpha, MIP-1 beta, RANTES and VEGF. Cytokine expression profiles were assessed on an EMD Millipore multiplex Luminex platform. Data from one patient who received reovirus is not included in this paper, as the majority of the Luminex output across all cytokines was below
the limit of detection. All data were normalized to respective pre-reovirus administration for each patient to serve as controls for the study.
Additionally the data from the 3 patients who did not receive reovirus were run through similar calculations to confirm that there was no change in cytokine expression over time. Thus the observed alterations in cytokine expression can be related to reovirus administration and not due to FOLFIRI and bevacizumab administration (Data not shown).
Biopsied specimens (from 1 patient who received reovirus: resected colostomy for pre reovirus sample; core biopsy for post reovirus sample) were fixed in neutral buffered formalin and subjected to paraffin embedding. The 5-µm-thick 10% formalin-fixed, paraffin-embedded tissue sections were deparaffinized in xylene three times, 10 minutes each, and subsequently rehydrated through graded alcohols to distilled water. Antigen heat retrieval was performed in 1 mM EDTA (pH 9.0) for 10 minutes (PMS2 15 minutes) using a microwave oven. Next, the sections were allowed to cool down in room temperature for 1.5 hours. After rinsing in distilled water and TBS successively, sections were incubated with rabbit polyclonal Granzyme B (Thermofisher Scientific # PA5-13518) at 4°C for 2 hours . Finally, the sections were covered with streptavidin peroxidase (Dako, Santa Barbara, CA) diluted 1:100 in PBS, incubated for 30 min at 37°C, washed three times in PBS and stained with 3,3’-diaminobenzidine as a substrate for the peroxidase for approximately 30 min at 37°C. Counter staining was performed using Mayer’s hematoxylin. Slides were scanned on the 3DHistech P250 slide scanner (SIG #1S10OD019961-01) using a 20X objective. Brown stain analysis was completed on the whole piece of tissue on every slide with 3DHistech Quant Center, using the DensitoQuant module. In this module, brown stain pixels were distinguished from the rest of the tissue by color thresh-holding. The analysis of blue versus brown areas
of tissue was completed in ImageJ, using the color threshold module. Finally, cells were viewed under 40X magnification in Case Viewer 2.3.
MICRO RNA ANALYSIS
For exosomal miRNA analysis, serum samples (from the 5 patients treated with reovirus and the 3 patients treated with background therapy only) were centrifuged at 12,500 G for 30 minutes. miRNA from 200 mL of supernatant was then extracted using Qiagen’s miRNeasy Serum/Plasma Advanced Kit (Cat#217204). For cellular miRNA analysis, PBMC cell pellets were lysed, and miRNA was extracted using Qiagen’s RNEasy FFPE kit (Cat#74404). 10 mg of total miRNA were analyzed (in quadruplicate for each sample) using miRNA assays 29a-3p, 26a-5p, 21-5p, 99-5p and 337-3p, obtained from ThermoFisher Scientific. RT-PCR quantification of miRNA assays for all samples was done using Applied Biosystems Taqman Advanced miRNA cDNA synthesis kit (Cat#A28007), with Cq analysis performed on Bio-RadCFX96 real time system C1000 touch thermal cycle.
Following total RNA isolation (from the 5 patients treated with reovirus and the 3 patients treated with background therapy only), single stranded cDNA synthesis was prepared by the method of fragmentation and labeling (ThermoFisher Scientific, Clariom D Pico Assay, human with arrays [Catalog Number 902924]). Briefly, total RNA was reverse transcribed using a reverse transcription priming method from an engineered set of primers that exclude sequences that match ribosomal RNA (rRNA). Thus, non- ribosomal RNA from the sample was primed (including both poly(A) and non-poly(A) mRNA) and converted into double-stranded cDNA using first and second strand enzymes from the said kit. Templates were used for in vitro transcription reactions at 37oC for 16 hours to yield cRNA. Next, hybridization cocktail from the hybridization kit (Catalog Number 900454) was used to generate ss-cDNA from cRNA
by the technique of chemical fragmentation followed by biotin labeling. Strictly adhering to the Affymetrix Genechip protocol, the ss-cDNA was hybridized to the Clariom D GeneChip probe array. The array image was finally generated by a high-resolution GeneArray Scanner 3000 7G (ThermoFisher Scientific, Santa Clara, CA,). For this study we analyzed 847 immune related genes (Supplementary Table
1) and compared the post reovirus treatment at days 2, 8 and 15 to the pre-reovirus treatment samples. Up or down regulation was reported for all significant changes (p<0.001) in expression, and those with a two- fold change in upregulation or half-fold change in down regulation were further analyzed.
For the transcriptome analysis, individual sample signals for each patient at each time point were extracted from the TAC (Transcriptome Assay Console: Thermofisher Scientific) software, organized and compiled in Microsoft Office Excel. Gene expression data were analyzed by the 2−ΔΔCT method  and normalized to the individual pre-reovirus (i.e., baseline) levels for each gene. Two tailed t-test was used to determine statistical significance (p<0.001). Statistics were calculated using Excel. For the miRNA and cytokine expression results, all patient samples for a given timepoint were pooled; baseline values were normalized to “1”, and the means for all subsequent timepoints were calculated relative to the baseline value. The standard error of the mean was calculated for each timepoint as well. Statistical significance was determined by using a paired t-test, and values less than or equal to 0.05 were reported.