An ISO is nothing more than a container. When an ISO is "burned" to any storage media the contents are extracted from ISO and stored on the media so in short being an ISO has nothing to do with booting.
Things that do matter
- The partition scheme (MBR vs GPT)
- The filesystem (NTFS should work but it's recommended to use fat32)
- The boot files available from the ISO
For UEFI boot (secure boot enabled or disabled) the partition scheme needs to be GPT and for legacy it needs to be MBR
On a GPT disk there is a fake MBR record for compatibility and I believe Rufus creates a real one for what they call a hybrid partition scheme (not officially supported)