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Preheat the oven to 375°F. Spray a large (9x5) loaf pan with cooking spray.
Melt the butter in a large skillet over medium heat. Add the diced onions, bell peppers, mushrooms, salt, and pepper. Sauté for 1-2 minutes before stirring, then let brown for another 1-2 minutes. Remove from heat and let cool for 5 minutes.
In a large bowl, blend the ketchup, Worcestershire sauce, and eggs until smooth. Add the ground beef, sautéed vegetables, and panko breadcrumbs, and mix thoroughly with your hands until well combined.
Press half of the meat mixture into the prepared loaf pan. Lay half of the provolone cheese slices in a single layer through the center of the meatloaf, about 2 inches wide. Top with the remaining meat mixture.
Bake for 40 minutes. Remove from the oven, top with the remaining provolone cheese slices, and return to the oven. Bake for an additional 15-20 minutes, until the cheese is melted and bubbly.
Let the meatloaf rest for 10 minutes before slicing and serving. Enjoy!