# Dissemination of Mitochondrial DNA Variants: Looking at the ‘Bigger’ Picture of the Tumour Microenvironment in Rectal Cancer Patients

## TL;DR

This study explores how mitochondrial DNA variants in extracellular vesicles relate to tumor aggressiveness in rectal cancer patients, using MRI and blood samples.

## Contribution

The study links low EV-mtDNA variant clearance to aggressive tumor features like high cell density and vascular invasion in rectal cancer.

## Key findings

- Low EV/WB-mtDNA TVN and HVN correlate with low ADC and large tumor volume in rectal cancer patients.
- Reduced EV-mtDNA variant clearance is associated with positive EMVI and higher histologic ypN stage.
- High TME cell density may hinder the removal of mutated mtDNA via EVs, promoting cancer aggressiveness.

## Abstract

The tumour microenvironment (TME) constitution is decisive for cancer outcome and is manifested in diffusion‐weighted (DW) magnetic resonance imaging (MRI). We hypothesized that the TME metabolic state is reflected by mitochondrial DNA (mtDNA) secreted in extracellular vesicles (EVs) and examined whether plasma EV‐mtDNA variants may divulge MRI‐assessed TME attributes of rectal cancer aggressiveness. On the diagnostic MRI scans from 60 rectal cancer patients, the apparent diffusion coefficient (ADC) was calculated on DW images (n = 29), and tumour volume (n = 57) and extramural vascular invasion (EMVI; all patients) were determined on anatomical images. Plasma EVs (all patients) were isolated by size exclusion chromatography and verified for EV features. The EV‐mtDNA was sequenced along with mtDNA in whole blood (WB; normal tissue) to calculate the EV/WB‐mtDNA total variant number (TVN) and heteroplasmic variant number (HVN)—as a proxy for TME intracellular mtDNA variants expelled in EVs. Low EV/WB‐mtDNA TVN and HVN, indicative of hampered clearance of mutated mtDNA via EVs, were associated with low ADC (high TME cell density; p = 0.018, p = 0.005) and a large tumour volume (p = 0.002, p = 0.003). Likewise, low EV/WB‐mtDNA TVN and HVN were associated with positive EMVI (tumour infiltration in blood vessels; p = 0.002, p = 0.003) and histologic ypN stage 1–2 (lymph nodes with tumour cells surviving radiotherapy; p = 0.002, p = 0.005), both indicators of high tumour aggressiveness. High cellular density may hamper the clearance of pathogenic tumour mtDNA variants by EVs and thus promote rectal cancer aggressiveness.

Trial Registration: ClinicalTrials.gov: NCT01816607. Registered 22 March 2013, https://clinicaltrials.gov/ct2/show/NCT01816607