Disclosed are mammalian tumor and/or cancer cell conditioned substrate preparations having tumor inhibiting activity. In some embodiments, the mammalian tumor and/or cancer cell conditioned substrate preparations are essentially free of cellular components. These preparations comprise a conditioned heterologous acellular collagenous tissue preparation, and may be prepared using a mammalian extracellular matrix substrate. The conditioned substrates include many different anti-tumor and/or anti-cancer biomolecules, such as that population of anti-tumor biomolecules that are secreted and/or produced by mammalian tumor and/or cancer cells as they grow on a substrate, thus imparting the anti-tumor and/or anti-cancer properties to the conditioned substrates of the invention. The present disclosure also provides methods for preparing the mammalian tumor and/or cancer cell conditioned substrates, as well as methods for using the preparations to inhibit tumor growth, such as in a vaccine or wound dressing. Methods for inhibiting prostate tumors and melanoma with the described conditioned substrates are also described. The conditioned mammalian tumor and/or cancer cell substrate preparations are essentially free of viable and/or carcinogenic or invasive tumor and/or cancer cells. The conditioned substrates and substrate preparations include anti-tumor and anti-cancer properties that may be used in preparations and formulations for the treatment of cancer.