DOI: 10.5061/dryad.g4f4qrfts
title: RNAseq transcriptome of draining lymph node (LN) and tumor of MC38 murine tumors treated with cryoablation and chitosan/IL-12

description: Focal ablation technologies are routinely used in the clinical management
 of inoperable solid tumors but often result in incomplete ablations
 leading to high recurrence rates. Adjuvant therapies capable of safely
 eliminating residual tumor cells are therefore of great clinical interest.
 Interleukin 12 (IL-12) is a potent antitumor cytokine that can be
 localized intratumorally through coformulation with viscous biopolymers
 including chitosan (CS) solutions. The objective of this research was to
 determine if localized immunotherapy with CS/IL-12 could prevent tumor
 recurrence after cryoablation (CA). Tumor recurrence, overall survival,
 and protective immunity were assessed. Systemic immunity was evaluated in
 spontaneously metastatic and bilateral tumor models. Temporal bulk RNA
 sequencing was performed on tumor and draining lymph node samples. In
 multiple murine tumor models, the addition of CS/IL-12 to CA reduced
 recurrence rates by 30–55%. Altogether, this cryo-immunotherapy induced
 complete durable regression of large tumors in 80–100% of treated animals.
 Mice treated with CA plus adjuvant CS/IL-12 were partially or completely
 protected from tumor rechallenge. Systemically, CS/IL-12 prevented lung
 metastases when delivered as a neoadjuvant to CA. However, CA plus
 CS/IL-12 had minimal antitumor activity against established, untreated
 abscopal tumors. Adjuvant anti-PD-1 therapy delayed the growth of abscopal
 tumors. Transcriptome analyses revealed early immunological changes in the
 dLN, followed by a significant increase in gene expression associated with
 immune suppression and regulation. Cryo-immunotherapy with localized
 CS/IL-12 reduces recurrences and enhances the elimination of large primary
 tumors. This focal combination therapy also induces significant systemic
 antitumor immunity although further studies are necessary.
description: MC38 tumors were implanted
 subcutaneously in the right flanks of female C57BL/6 mice as described
 above. When tumor volumes measured between 200–500
 mm<sup>3</sup>, CA was performed and CS/IL-12 was
 intratumorally injected within an hour after CA. Mice were euthanized 3
 days and 7 days post-ablation to harvest tumor tissues and tumor-draining
 inguinal lymph nodes (dLNs). To obtain cell suspensions, dLNs were
 homogenized through 40 µm mesh strainers with complete T cell media. Tumor
 tissues were first minced into approximately 0.5 cm squares before
 treatment with 5X Triple Enzyme Solution (collagenase type IV 10 mg/ml,
 hyaluronidase 1 mg/ml, and DNase 20,000 units final concentration in
 Hank’s Buffered Saline Solution) for 30 minutes at 37°C with magnetic bar
 stirring. The resulting homogenate was filtered through a 70 µm mesh
 strainer into a clean tube to achieve the final cell suspension.
 Total RNA was extracted from the cell
 suspension using the GeneJET RNA Purification Kit (ThermoFisher cat
 #K0731) according to the manufacturer’s protocol. Briefly, Lysis Buffer
 supplemented with β-mercaptoethanol and then ethanol (99%) was added to
 the cell suspension and vortexed. The lysate was then loaded onto the
 purification spin column and washed twice before eluting with
 nuclease-free water. Samples were sent to BGI Genomics (Hong Kong, China)
 to perform transcriptome sequencing using their
 DNBSEQ<sup>TM</sup> platform using a read length of 100
 paired-end base pairs. Data analysis was performed using the BGI software
 platform, Dr. Tom. Sample
 inclusion was based on RNA quality (260/280 &gt; 1.8) and successful
 library construction. Only one sample, from a tumor treated with CA +
 CS/IL-12 (11_T_D3), failed to meet these criteria and was omitted from
 further analysis. The dLN from one mouse treated with CA alone and then
 euthanized on day 7 after treatment was not recovered during necropsy
 (34_dLN_D7). Secondary exclusion criteria were based on read alignment
 coverage and randomness. The technical difficulty of extracting
 high-quality RNA from highly necrotic ablated tumor samples, in particular
 three days after ablation, hindered the interpretation of gene expression
 from the tumor at day 3 post-treatment. Unless otherwise noted, for all gene
 expression analysis, genes were filtered from the transcriptome using a
 gate of Q &lt; 0.05 for the comparisons of interest. Bowtie2 was used
 to map the clean reads to the reference gene sequence (transcriptome), and
 then RSEM was used to calculate the gene expression level of each sample.
 Differential gene detection was calculated using the DESeq2 method, where
 Q is calculated using the FDR/Benjamini-Hochberg method. According to the
 results of differential gene detection, the R package pheatmap was used to
 perform hierarchical clustering analysis on the union set differential
 genes.
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