Enhanced plant-derived vesicles for nucleotide delivery for cancer therapy.

Small RNAs (microRNAs [miRNAs] or small interfering RNAs [siRNAs]) are effective tools for cancer therapy, but many of the existing carriers for their delivery are limited by low bioavailability, insufficient loading, impaired transport across biological barriers, and low delivery into the tumor microenvironment. Extracellular vesicle (EV)-based communication in mammalian and plant systems is important for many physiological and pathological processes, and EVs show promise as carriers for RNA interference molecules. However, some fundamental issues limit their use, such as insufficient cargo loading and low potential for scaling production. Plant-derived vesicles (PDVs) are membrane-coated vesicles released in the apoplastic fluid of plants that contain biomolecules that play a role in several biological mechanisms. Here, we developed an alternative approach to deliver miRNA for cancer therapy using PDVs. We isolated vesicles from watermelon and formulated a hybrid, exosomal, polymeric system in which PDVs were combined with a dendrimer bound to miRNA146 mimic. Third generation PAMAM was chosen due to its high branching structure and versatility for loading molecules of interest. We performed several in vivo experiments to demonstrate the therapeutic efficacy of our compound and explored in vitro biological mechanisms underlying the anti-tumor effects of miRNA146, which are mostly related to its anti-angiogenic activity.

A Th2-score in the tumor microenvironment as a predictive biomarker of response to Bacillus Calmette Guérin in patients with non-muscle invasive bladder carcinoma: A retrospective study.

Intravesical Bacillus Calmette Guerin (BCG) is the gold standard therapy for intermediate/high-risk non-muscle invasive bladder cancer (NMIBC). However, the response rate is ~60%, and 50% of non-responders will progress to muscle-invasive disease. BCG induces massive local infiltration of inflammatory cells (Th1) and ultimately cytotoxic tumor elimination. We searched for predictive biomarker of BCG response by analyzing tumor-infiltrating lymphocyte (TIL) polarization in the tumor microenvironment (TME) in pre-treatment biopsies. Pre-treatment biopsies from patients with NMIBC who received adequate intravesical instillation of BCG (n = 32) were evaluated retrospectively by immunohistochemistry. TME polarization was assessed by quantifying the T-Bet+ (Th1) and GATA-3+ (Th2) lymphocyte ratio (G/T), and the density and degranulation of EPX+ eosinophils. In addition, PD-1/PD-L1 staining was quantified. The results correlated with BCG response. In most non-responders, Th1/Th2 markers were compared in pre-and post-BCG biopsies. ORR was 65.6% in the study population. BCG responders had a higher G/T ratio and a greater number of degranulated EPX+ cells. Variables combined into a Th2-score showed a significant association with higher scores in responders (p = 0.027). A Th2-score cut-off value >48.1 allowed discrimination of responders with 91% sensitivity but lower specificity. Relapse-free survival was significantly associated with the Th2-score (p = 0.007). In post-BCG biopsies from recurring patients, TILs increased Th2-polarization, probably reflecting BCG failure to induce a pro-inflammatory status and, thus, a lack of response. PD-L1/PD-1 expression was not associated with the response to BCG. Our results support the hypothesis that a pre-existing Th2-polarized TME predicts a better response to BCG, assuming a reversion to Th1 polarization and antitumor activity.

Implications of Guideline-based, Risk-stratified Restaging Transurethral Resection of High-grade Ta Urothelial Carcinoma on Bacillus Calmette-Guérin Therapy Outcomes.

BACKGROUND: Guideline indications for restaging transurethral resection (reTUR) for high-grade (HG) Ta bladder tumors vary due to a paucity of data. OBJECTIVE: To investigate guideline-based, risk-adapted approaches to reTUR for HG Ta lesions. DESIGN, SETTING, AND PARTICIPANTS: An institutional review of HG Ta patients who received adequate bacillus Calmette-Guérin (BCG) from 2000 to 2019 was conducted. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Guideline criteria for reTUR were used to stratify patients. Kaplan-Meier product limits estimated survival. Cox regression and log-rank tests identified association of variables with survival. RESULTS AND LIMITATIONS: Of the 209 patients with HG Ta bladder cancer, 104 (50%) underwent reTUR, which identified residual disease in 39 patients (38%). Only one patient (1%) was upstaged to pT1 on reTUR. In all unstratified HG Ta patients, reTUR was associated with improved progression-free survival (p = 0.050) and recurrence-free survival (RFS; p = 0.003). The 5-yr RFS for patients who underwent versus those who did not undergo reTUR based on AUA guidelines was 73% (95% confidence interval 63-81%) versus 52% (40-62%), and for those who underwent versus those who did not undergo reTUR based on EAU guidelines was 76% (61-86%) versus 22% (4-49%). In 45 patients meeting both AUA high-risk criteria (large, multifocal tumors) and EAU criteria (lack of detrusor muscle) for reTUR, lack of restaging was associated with over a two-fold increase in recurrence (67% vs 15%, p = 0.002) and progression (25% vs 6%, p = 0.109). Data were limited by selection bias unaccounted for in selecting candidates for reTUR. CONCLUSIONS: Restaging TUR in all HG Ta patients, regardless of risk stratification, was associated with improved outcomes. The benefit of reTUR was most notable in high-risk patients without muscle in the index specimen, consistent with components of both AUA and EAU guidelines. These data support a non-risk-adapted approach to reTUR for all HG Ta lesions. PATIENT SUMMARY: Restaging bladder tumor resection improves outcomes in patients with high-grade Ta tumors treated with bacillus Calmette-Guérin (BCG).

Bacillus Calmette-Guérin Retains Clinically Relevant Viability for up to 72 Hours After Reconstitution: Potential Implications for Clinical Practice in Times of Shortage.

There have been repeated supply shortages of bacillus Calmette-Guérin (BCG), the gold-standard immunotherapy for patients with high-grade non-muscle-invasive bladder cancer (NMIBC). Organizations have issued guidance on coping with this shortage, including administering split-dose BCG such that one vial may treat up to three patients. However, logistical implementation of this strategy in a real-world setting is hampered by the recommendation to use BCG within 2 h of reconstitution. We assessed BCG viability in terms of colony-forming units (CFUs) and demonstrated that viability remained constant for at least 8 h after reconstitution (decline at 8 h of 9.1% for lot 1 [p = 0.3] and 4.8% for lot 2 [p = 0.2]). While the viability at 24 h was lower, it did not drop to a level below that of reducing the BCG dose to one-third (67% for lot 1 and 60% for lot 2) and remained close to 50% for at least 72 h. An in vitro model using co-culture of BCG and leukocytes with a BCG-sensitive cell line (RT4-V6) demonstrated no decrease in the cytotoxic potential of BCG at 72 h. In times of shortage, BCG vials may be split and administered for up to at least 8 h (or even 72 h) after reconstitution, allowing more patients to benefit from BCG while placing less strain on the logistics of clinical practice. PATIENT SUMMARY: The current supply of and increased demand for bacillus Calmette-Guérin (BCG), used in the treatment of bladder cancer, have led to repeated BCG shortages. One way to address this is to provide a reduced BCG dose to allow more patients to be treated. In this study we found that BCG viability remains clinically relevant up to 72 h after reconstitution, thus allowing for more patients to be treated from a single vial.

The obesity paradox: defining the impact of body mass index and diabetes mellitus for patients with non-muscle-invasive bladder cancer treated with bacillus Calmette-Guérin.

OBJECTIVES: To evaluate if the obesity paradox, wherein obesity portends worse overall prognosis for a disease but improved outcomes for patients receiving immunotherapy, exists for patients receiving bacillus Calmette-Guérin (BCG) in a contemporary cohort. PATIENTS AND METHODS: We performed an Institutional Review Board-approved database review to identify patients with non-muscle-invasive bladder cancer (NMIBC) completing at least an induction course of BCG. Clinicopathological variables collected included: body mass index (BMI), medications, and diabetes mellitus (DM). Outcomes of interest included: recurrence-free (RFS), progression-free (PFS), cancer-specific (CSS), and overall survival (OS). Univariate and multivariate modelling were used to evaluate the association between outcomes and clinical factors. RESULTS: A total of 579 patients (median follow-up 4.6 years) received BCG induction for NMIBC; 90% had high-grade disease (47.2% clinical stage T1). In all, 75.7% of patients were overweight or obese and 18% had DM. Aspirin, statins, metformin and ß-blockers were used in 34%, 42%, 11%, and 29% of patients, respectively. Overweight and obese patients had improved PFS, CSS and OS. DM was associated with worse RFS. Medications of interest had no association with outcomes. CONCLUSION: Elevated BMI is associated with improved outcomes in patients with NMIBC treated with BCG immunotherapy. Patients with DM are at increased risk of recurrence. These findings support a potential obesity paradox in bladder cancer. Evaluation of the underlying mechanism and the role of global patient assessment, counselling, and risk factor modification are warranted.

Poly(N-isopropylacrylamide)-based dual-crosslinking biohybrid injectable hydrogels for vascularization.

Injectable hydrogels provide a powerful and non-invasive approach for numerous applications in cell transplantation, growth factor delivery, tissue regeneration and so forth. The properties of injectable hydrogels should be well-tuned for specific applications, where their overall design should ensure biocompatibility, non-toxicity, robust mechanical properties, and most importantly the ability to promote vascularization and integration with the host tissue/organ. Among these criteria, vascularization remains a key design element in the development of functional therapeutic hydrogels for successful translation into clinical settings. To that end, there is still a critical need for the development of the next generation of injectable hydrogels with precisely tuned biophysical and biochemical properties which could simultaneously promote tissue vascularization. In this work, we developed a temperature responsive, dual-crosslinking, biohybrid hydrogels, modified with a vasculogenic peptide for applications in regenerative medicine, specifically tissue vascularization. The synthesized hydrogels consisted of poly(N-isopropylacrylamide)-based copolymer, functionalized gelation and angiogenic VEGF-mimetic QK peptide with enhanced shear-thinning and injectability properties. QK peptide is a VEGF-mimetic vasculogenic peptide which binds to VEGF receptors and activates intercellular pathway for vascularization. Apart from the presence of QK peptide, the mechanical properties of the hydrogels were precisely tuned by altering the polymer concentration, enabling successful assembly and endothelial cell network formation. Extended in vitro studies demonstrated successful encapsulation and homogeneous distribution of endothelial cells within the three-dimensional (3D) environment of the hydrogel matrix with significantly enhanced vascularization in presence of the QK peptide as early as 3 days of culture. A small, preliminary in vivo study in mice showed a trend of increased blood vessel formation in hydrogels that incorporated the QK peptide. Overall, our study presents the design and characterization of injectable, dual-crosslinking and vasculogenic hydrogels with controlled properties which could be utilized for numerous applications in regenerative medicine, minimally invasive cell and drug delivery as well as fundamental studies on tissue vascularization and angiogenesis. STATEMENT OF SIGNIFICANCE: In this work, we synthesized a new class of temperature responsive, dual-crosslinking, biohybrid injectable hydrogels with enhanced vascularization properties for broad applications in regenerative medicine and minimally invasive cell/drug delivery. The developed hydrogels properly accommodated 3D culture, assembly and network formation of endothelial cells, as evidenced by in vitro and in vivo studies.

Multi-Institution Evaluation of Sequential Gemcitabine and Docetaxel as Rescue Therapy for Nonmuscle Invasive Bladder Cancer.

PURPOSE: Rescue intravesical therapies for patients with bacillus Calmette-Guérin failure nonmuscle invasive bladder cancer remain a critical focus of ongoing research. Sequential intravesical gemcitabine and docetaxel therapy has shown safety and efficacy in 2 retrospective, single institution cohorts. This doublet has since been adopted as an intravesical salvage option at multiple institutions. We report the results of a multi-institutional evaluation of gemcitabine and docetaxel. MATERIALS AND METHODS: Each institution retrospectively reviewed all records of patients treated with intravesical gemcitabine and docetaxel for nonmuscle invasive bladder cancer between June 2009 and May 2018. Only patients with recurrent nonmuscle invasive bladder cancer and a history of bacillus Calmette-Guérin treatment were included in the analysis. If patients were disease-free after induction, maintenance was instituted at the treating physician's discretion. Posttreatment surveillance followed American Urological Association guidelines. Survival analysis was performed using the Kaplan-Meier method and risk factors for treatment failure were assessed with Cox regression models. RESULTS: Overall 276 patients (median age 73 years, median followup 22.9 months) received treatment. Nine patients were unable to tolerate a full induction course. One and 2-year recurrence-free survival rates were 60% and 46%, and high grade recurrence-free survival rates were 65% and 52%, respectively. Ten patients (3.6%) had disease progression on transurethral resection. Forty-three patients (15.6%) went on to cystectomy (median 11.3 months from induction), of whom 11 (4.0%) had progression to muscle invasion. Analysis identified no patient, disease or prior treatment related factors associated with gemcitabine and docetaxel failure. CONCLUSIONS: Intravesical gemcitabine and docetaxel therapy is well tolerated and effective, providing a durable response in patients with recurrent nonmuscle invasive bladder cancer after bacillus Calmette-Guérin therapy. Further prospective study is warranted.

Microfluidic Tumor-Vascular Model to Study Breast Cancer Cell Invasion and Intravasation.

Cancer is a major leading cause of disease-related death in the world. The severe impact of cancer can be attributed to poor understanding of the mechanisms involved in earliest steps of the metastatic cascade, specifically invasion into the surrounding stroma and intravasation into the blood capillaries. However, conducting integrated biological studies of invasion and intravasation have been challenging, within in vivo models and traditional in vitro assay, due to difficulties in establishing a precise tumor microenvironment. To that end, in this work, a novel 3D microfluidic platform comprised of concentric three-layer cell-laden hydrogels for simultaneous investigation of breast cancer cell invasion and intravasation as well as vasculature maturation influenced by tumor-vascular crosstalk is developed. It was demonstrated that the presence of spontaneously formed vasculature enhance MDA-MB-231 invasion into the 3D stroma. Following invasion, cancer cells are visualized intravasating into the outer vasculature. Additionally, invading cancer cells significantly reduce vessel diameter while increasing permeability, consistent with previous in vivo studies. Major signaling cytokines involved in tumor-vascular crosstalk that govern cancer cell invasion and intravasation are further identified. Taken together, this platform will enable unique insights of critical biological events within the metastatic cascade, with significant potential for developing efficient cancer therapeutics.