Love that phrase "of course". Can't let him think it's okay to have doubts or insecurities.
Oh. Is that sarcasm?
Little bit. But it's also a nice way to show that Twilight's still a long way from a perfect parent, and Spike loves her anyway. (Not sure why you asked that if you were going to close the comments so I couldn't reply.)
I do get he was confused about what exactly his relationship should be but that Twilight cares about him never seemed to be in doubt. It is more rather he could consider him her son. However even if she does not consider herself his mother that doesn't mean she doesn't care about him.
Yet this is exactly what Sombra did with Spike. He took his doubts regarding his relationship with Twilight,slowly changed the narration and made Spike believe she didn't care. Having a direct access to Spike's mind and manipulating his dragon greed was why he (almost) succeeded.
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I love this panel. Twilight's face on the third frame is so sincere and loving and worried. This is exactly why I love Twilight and Spike. Sorry that I don't have more to say other then there's something about this dialog and these emotions that is just tear jerking. I'm very excited to see how it all ends.