"Spider-Man: Across the Spider-Verse" doesn't explicitly label Gwen Stacy as transgender, but there are strong hints to that effect. Trans art, often created under oppressive conditions, has a long history of explicating its ideas through thinly veiled subtext. For example, although "The Matrix" stars Keanu Reeves as a cisgender, heterosexual man, it was created by two trans women who have loudly confirmed what film theorists long suspected; that the movie is an allegory for the trans experience (via NPR).
But "Across the Spider-Verse" is much clearer in its depiction of Gwen's trans identity than the Wachowski sisters could afford to be in 1999. Not only does Gwen have a trans pride flag embossed with the words "PROTECT TRANS KIDS" hanging over her bedroom door, but her universe of origin is consistently rendered in shades of white, blue, and pink, reflective of the trans pride flag colors.
As Gwen re-enters her universe of origin and finally has a heart-to-heart with her dad, the character herself is entirely rendered in those colors, with even her hair taking on hues of the trans pride flag. In that same scene, her father can be seen wearing a trans pride patch above his police badge. And it should be noted that, as Gwen pours her heart out to her estranged dad, she tells him that she's struggled with hiding a part of herself from him. As @Blankzilla wrote on Twitter, "Being draped in the trans colors while giving a speech about having to hide half of yourself from the people you love is as subtle as a brick."
