Identification of immune-related genes prognostic index for predicting survival and immunotherapy in colorectal carcinoma

1.Clinical Data
Transcript data and Clinicopathological information was downloaded from The
Cancer Genome Atlas (TCGA) database (https://portal.Gdc.cancer.gov/), including 41 cases of para-tumor, 473 cases of CRC tumor and 452 clinical
cases.

The survival and transcriptional data of 250 CRC cases were downloaded from
the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The transcript dataset
GSE161158 uploaded in November 2020 by Moffitt Cancer Research Center,
University of Miami was used (10). Lists of immune related genes were download from ImmPort (https://www.immport. org/home) and Innate DB
(https://www.innatedb.ca/). KEGG (http://www.gsea-msigdb.org/gsea/index.jsp) gene
sets and all Gene Ontology (GO) gene sets were used as Gene Symbols. Gene
mutation information was downloaded from cBioPortal (http://www.cbioportal.org/).

2.Murine Data
2.1 IRGPI Genes Expression in CRC Murine Model
SPF Balb/c male mice, 6 - 8 weeks old, body mass (20 ± 5) g, purchased from
Huaxing Experimental Animal Farm of Huiji District (Zhengzhou City, China), experimental animal license NO. is SCXK (Yu) 2019-0002. All animal
experiments was approved by the Experimental Mouse Ethics Committee of Nanjing
University of Traditional Chinese Medicine (NO. 202010A026).
The above mice were randomly divided into: Control group (C), CRC Model
group (M)，10 per group. Five Balb/c male mice
were taken as tumor-bearing mice, and 1×107 CT26 cells were
subcutaneously injected into the left axilla, and sacrificed one week later. The
subcutaneous tumor was removed under sterile conditions, placed in sterile PBS,
and disintegrated into several 1 mm3 masses. Under sterile conditions,
the two groups of mice were dissected to expose the colon, the 1 mm3
tumor mass was fixed to the colon of the CRC Model group with tissue glue,
while nothing was fixed in the Control group, and then the abdomen of the two
groups of mice was sutured. After 3 days of postoperative recovery, mice were
weighed, and Micro-CT scans were performed on the 26th day (under the condition
of isoflurane respiratory anesthesia), and on the 27th day, the mice were
sacrificed after anesthesia with 2 % sodium pentobarbital. Total RNA was
extracted from the colon of the Control group and the tumor tissue of the CRC
Model group by FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme, China,
Cat#RC101-01) , and after reverse transcribed into cDNA using HiScript® Ⅲ RT SuperMix for q PCR (Vazyme, China, Cat#R323-01), Real-Time PCR was used
to detect the expression of IRCPI genes in each group
using BlastaqTM Green 2× qPCR MasterMix (abm, Canada, Cat#G891). The primer sequences are shown in Supplementary
Table S4.
2.2 Immune Infiltration in CRC
murine model
The liver, colon, tumor, and mesentery of paraffin-embedded mice were
sectioned, and then stained with hematoxylin-eosin (HE staining), and
photographed with an upright white light photographic microscope (Nikon, Japan,
Eclipse Ci-L).

TIME immune cells were detected by flow cytometry (FCM). PBMCs were
extracted by RBC lysate (FcMACS, China, Cat#FMS-RBC500). At least 5×106
cell suspensions(100 μL) were incubated with FC blocker at 4 ℃ for 10 min, then
Anti-Human/Mouse CD11b FITC Antibody (PeproTech, USA, Cat#03221-50)、PE-Cy™7 Rat Anti-Mouse CD86 Antibody (BD Pharmingen™, USA, Cat#560582) and
Alexa Fluor® 488 Anti-Mouse CD206 Antibody (Biolegend, USA, Cat#141710) were used
to marked Macrophages; Alexa Fluor® 488 anti-mouse CD19 Antibody (Invitrogen,
USA, REF#11-0193-81) and PE/Cy7 anti-mouse/rat/human CD27 Antibody (Biolegend,
USA, Cat#124216) were used to marked B cells; Anti-Mouse CD4 APC-Cyanine7
(PeproTech, USA, Cat#06122-87)、Anti-Mouse CD8a FITC Antibody (PeproTech, USA, Cat#10122-50)、Anti-Mouse CD25 APC Antibody (PeproTech, USA, Cat#07312-80) and
Anti-Mouse/Rat FOXP3 PE Antibody (PeproTech, USA, Cat#83422-60) were used to
marked T cells, and PBMCs monochromic tubes were made respectively. The cells
were detected on the Amnis FlowSight flow cytometer (Merck Millipore, USA), and
immunocyte subsets were analyzed using the IDEAS software (Merck Millipore,
USA). Supplement Fig.S7 visualized the analysis strategies for IRGPI
immunocyte subsets by Flow cytometry.