The
Spider Rune will likely be the last rune you unlock (apart from the one automatically unlocked by completing the game). The Vine, Teleportation, and Ram runes are all required before going for this. It is earned by going from
The Prisoner's Quarters (first area) and exiting through to
The Toxic Sewers, then exit to
The Ancient Sewers. through to
The Insufferable Crypt to face the
Conjuctivius boss. You should be able to exit this boss battle into a new area called
The Slumbering Sanctuary; a very tough area with invisible enemies.
As you work your way through
The Slumbering Sanctuary, you will reach a large pit, with a red boss banner. Dropping down with commence a fight with and Elite Orb Caster, which you will need to defeat. Killing them will drop the
Spider Rune, automatically equipped as a permanent unlock. This fight can be quite tough, but dodge rolling is extremely important.
With the
Spider Rune equipped, you will be able to wall climb and wall jump to make progress through the game and give access to new and secret areas. It will also allow us to unlock new areas like the
Prison Depths accessed from
Promenade of the Condemned, and the
Graveyard from the
Conjuctivius boss battle.