Plasma-Derived Cell-Free DNA as a Biomarker for Early Detection, Prognostication, and Personalized Treatment of Urothelial Carcinoma.

Bladder cancer (BC) is one of the most common malignancies in the United States, with over 80,000 new cases and 16,000 deaths each year. Urothelial carcinoma (UC) is the most common histology and accounts for 90% of cases. BC management is complicated by recurrence rates of over 50% in both muscle-invasive and non-muscle-invasive bladder cancer. As such, the American Urological Association (AUA) recommends that patients undergo close surveillance during and after treatment. This surveillance is in the form of cystoscopy or imaging tests, which can be invasive and costly tests. Considering this, there have been recent pushes to find complements to bladder cancer surveillance. Cell-free DNA (CfDNA), or DNA released from dying cells, and circulating tumor DNA (ctDNA), or mutated DNA released from tumor cells, can be analyzed to detect and characterize the molecular characteristics of tumors. Research has shown promising results for ctDNA use in the BC care realm. A PubMed literature review was performed finding studies discussing cfDNA and ctDNA in BC detection, prognostication, and monitoring for recurrence. Keywords used included bladder cancer, cell-free DNA, circulating tumor DNA, urothelial carcinoma, and liquid biopsy. Studies show that ctDNA can serve as prognostic indicators of both early- and late-stage BC, aid in risk stratification prior to major surgery, assist in detection of disease progression and metastatic relapse, and can assess patients who may respond to immunotherapy. The benefit of ctDNA is not confined to BC, as studies have also suggested its promise as a biomarker for neoadjuvant chemotherapy in upper-tract UC. However, there are some limitations to ctDNA that require improvements in ctDNA-specific detection methods and BC-specific mutations before widespread utilization can be achieved. Further prospective, randomized trials are needed to elucidate the true potential ctDNA has in advancements in BC care.

The M2a Macrophage Phenotype Accompanies Pulmonary Granuloma Resolution in Mmp12 Knock-Out Mice Instilled with Multiwall Carbon Nanotubes.

Sarcoidosis is a chronic disease with unknown etiology and pathophysiology, characterized by granuloma formation. Matrix Metalloproteinase-12 (MMP12) is an elastase implicated in active granulomatous sarcoidosis. Previously, we reported that oropharyngeal instillation of multiwall carbon nanotubes (MWCNT) into C57Bl/6 mice induced sarcoid-like granulomas and upregulation of MMP12. When Mmp12 knock-out (KO) mice were instilled with MWCNT, granuloma formation occurred 10 days post-instillation but subsequently resolved at 60 days. Thus, we concluded that MMP12 was essential to granuloma persistence. The aim of the current study was to identify potential mechanisms of granuloma resolution in Mmp12KO mice. Strikingly, an M2 macrophage phenotype was present in Mmp12KO but not in C57Bl/6 mice. Between 10 and 60 days, macrophage populations in MWCNT-instilled Mmp12KO mice demonstrated an M2c to M2a phenotypic shift, with elevations in levels of IL-13, an M2 subtype-regulating factor. Furthermore, the M2 inducer, Apolipoprotein E (ApoE), and Matrix Metalloproteinase-14 (MMP14), a promoter of collagen degradation, were upregulated in 60-day MWCNT-instilled Mmp12KO mice. In conclusion, alveolar macrophages express two M2 phenotypes in Mmp12KO mice: M2c at 10 days when granulomas form, and M2a at 60 days when granulomas are resolving. Findings suggest that granuloma resolution in 60-day Mmp12KO mice requires an M2a macrophage phenotype.

Myeloid ABCG1 Deficiency Enhances Apoptosis and Initiates Efferocytosis in Bronchoalveolar Lavage Cells of Murine Multi-Walled Carbon Nanotube-Induced Granuloma Model.

The use of carbon nanotubes has increased in the past few decades. Carbon nanotubes are implicated in the pathogenesis of pulmonary sarcoidosis, a chronic granulomatous inflammatory condition. We developed a murine model of chronic granulomatous inflammation using multiwall carbon nanotubes (MWCNT) to investigate mechanisms of granuloma formation. Using this model, we demonstrated that myeloid deficiency of ATP-binding cassette (ABC) cholesterol transporter (ABCG1) promotes granuloma formation and fibrosis with MWCNT instillation; however, the mechanism remains unclear. Our previous studies showed that MWCNT induced apoptosis in bronchoalveolar lavage (BAL) cells of wild-type (C57BL/6) mice. Given that continual apoptosis causes persistent severe lung inflammation, we hypothesized that ABCG1 deficiency would increase MWCNT-induced apoptosis thereby promoting granulomatous inflammation and fibrosis. To test our hypothesis, we utilized myeloid-specific ABCG1 knockout (ABCG1 KO) mice. Our results demonstrate that MWCNT instillation enhances pulmonary fibrosis in ABCG1 KO mice compared to wild-type controls. Enhanced fibrosis is indicated by increased trichrome staining and transforming growth factor-beta (TGF-ß) expression in lungs, together with an increased expression of TGF-ß related signaling molecules, interleukin-13 (IL-13) and Smad-3. MWCNT induced more apoptosis in BAL cells of ABCG1 KO mice. Initiation of apoptosis is most likely mediated by the extrinsic pathway since caspase 8 activity and Fas expression are significantly higher in MWCNT instilled ABCG1 KO mice compared to the wild type. In addition, TUNEL staining shows that ABCG1 KO mice instilled with MWCNT have a higher percentage of TUNEL positive BAL cells and more efferocytosis than the WT control. Furthermore, BAL cells of ABCG1 KO mice instilled with MWCNT exhibit an increase in efferocytosis markers, milk fat globule-EGF factor 8 (MFG-E8) and integrin ß3. Therefore, our observations suggest that ABCG1 deficiency promotes pulmonary fibrosis by MWCNT, and this effect may be due to an increase in apoptosis and efferocytosis in BAL cells.