Variations in Circulating Tumor Microenvironment-Associated Proteins in Non-Muscle Invasive Bladder Cancer Induced by Mitomycin C Treatment

Abstract

Mitomycin C (MMC) is a widely employed chemotherapeutic agent, particularly in non-muscle invasive bladder cancer (NMIBC), where it functions by inducing DNA cross-linking and promoting tumor cell apoptosis. However, the tumor microenvironment (TME) significantly influences the therapeutic efficacy of MMC. Among the key regulators within the TME, the complement system and the coagulation pathway play a crucial role in modulating immune responses to cancer therapies, including MMC. This article explores the interaction between platinum nanoparticles (PtNPs) with human serum (HS) of NMIBC patients (T1 and Ta subtypes) at three different points: before the chemotherapy instillation of MMC (t₀) and three (t₃) and six months (t₆) after the treatment with MMC. This novel nanoproteomic strategy allowed the identification of a TME proteomic signature associated with the response to MMC treatment. Importantly, two proteins involved in the immune response were found to be deregulated across all patients (T1 and Ta subtypes) during MMC treatment: prothrombin (F2) downregulated and complement component C7 (C7) upregulated. By understanding how these biomarker proteins interact with MMC treatment, novel therapeutic strategies can be developed to enhance treatment outcomes and overcome resistance in NMIBC.

Keywords: SWATH-MS; complement system; mitomycin C (MMC); non-muscle invasive bladder cancer (NMIBC); platinum nanoparticles (PtNPs); protein corona (PC); tumor microenvironment (TME).

Figure 1

Diagrammatic overview of the protocol for PC assembly on PtNPs (2.40 ± 0.30 nm), following ex vivo incubation with HS from n = 42 healthy controls (HCs) and n = 42 NMIBC patients. Samples were collected at baseline (t₀, pre-treatment) and three (t₃) and six months (t₆) post-mitomycin C (MMC) instillation.

Figure 2

Flowchart depicting the general pretreatment, depletion with DDT, and alkylation with IAA of human serum samples before the incubation with PtNPs (2.40 ± 0.30 nm) for the PC formation.

Figure 3

Venn diagram depicting the overlap and subtype-specific differentially expressed serum proteins adsorbed onto PtNPs (2.40 ± 0.30 nm) after 30 min ex vivo incubation with HS from NMIBC patients of subtypes T1 and Ta (data for the T1 subtype are highlighted in blue color and for the Ta subtype in orange).

Figure 4

Venn diagram showing the number of shared and specific deregulated proteins identified in the PC-coated PtNPs (2.40 ± 0.30 nm) after their incubation (30 min) with HS samples from NMIBC patients of the T1 subtype (left) and Ta subtype (right) at different times (t₀, t₃, and t₆).

Figure 5

Clusters found in the protein–protein interaction network map based on the STRING database, highlighting proteins associated with the immune response pathway. At the top, clusters with 13, 17, and 17 deregulated proteins related to the immune response pathway found in the T1 subtype at t₀, t₃, and t₆, respectively, are shown. A total of 23, 12, and 22 proteins related to the immune response pathway were deregulated in the Ta subtype at times t₀, t₃, and t₆, respectively (down).

Figure 6

Synthesis of citrate-coated PtNPs (2.40 ± 0.30 nm).