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The significance of lactate transporters has been recognized in various cancer types, but their role in urothelial carcinoma remains mostly unknown. The aim of this study was to investigate the functional importance of the monocarboxylate transporter (MCT) 4 in preclinical models of urothelial carcinoma and to assess its relevance in patient tumors. The association of MCT4 expression with molecular subtypes and outcome was determined in The Cancer Genome Atlas (TCGA) cohort and two independent cohorts of patients with urothelial carcinoma. Silencing of MCT4 was performed using siRNAs in urothelial carcinoma cell lines. Effects of MCT4 inhibition on cell growth, apoptosis, and production of reactive oxygen species (ROS) were assessed. Moreover, effects on lactate efflux were determined. The in vivo effects of MCT4 silencing were assessed in an orthotopic xenograft model. MCT4 expression was higher in the basal subtype. Decreased MCT4 methylation and increased RNA and protein expression were associated with worse overall survival (OS). Inhibition of MCT4 led to a reduction in cell growth, induction of apoptosis, and an increased synthesis of ROS. MCT4 inhibition resulted in intracellular accumulation of lactate. In vivo, stable knockdown of MCT4 reduced tumor growth. The expression of MCT4 in urothelial carcinoma is associated with features of aggressive tumor biology and portends a poor prognosis. Inhibition of MCT4 results in decreased tumor growth in vitro and in vivo. Targeting lactate metabolism via MCT4 therefore provides a promising therapeutic approach for invasive urothelial carcinoma, especially in the basal subtype.

Suppl. Table 1: Characteristics of the TCGA cohort; Suppl. Table 2: Characteristics of cohort A. CIS= carcinoma in situ; Suppl. Table 3: Characteristics of cohort B Suppl. Fig. 1. Correlation of degree of DNA methylation at CpG site cg10183885 in the SLC16A3 gene region (see Suppl. Table 4) and MCT4 mRNA expression; Suppl. Fig. 2. Correlation of gene expression and protein expression in cohort B. Suppl. Fig. 3. Representative H&E (upper panel) and immunohistochemical images in matched bladder cancer samples (each column is one patient). CD8 was used to stain T cells (middle panel) in comparison with MCT4 expression (lower panel). Suppl. Fig. 4. Protein expression of MCT4 in benign urothelium, primary tumor tissue and lymph node metastases (cohort A). Suppl. Fig. 5. Protein expression in primary tumors according to tumor stage (left) and lymph node involvement (right) (cohort A).; Suppl. Fig. 6. MCT4 mRNA expression correlates with molecular subtypes in the TCGA cohort using the updated TCGA nomenclature (n=408); Suppl. Fig. 7. MCT4 gene expression does not correlate with overall survival in a cohort of patients undergoing neoadjuvant chemotherapy; Suppl. Fig. 8. Expression of MCT4 in urothelial carcinoma cell lines detected by Western Blot (A) and qPCR (B); Suppl. Fig. 9. Effect of MCT4 silencing on cell growth in hypoxic conditions determined by MTT assay. Suppl. Fig. 10. Effect of inhibition of MCT4 using siRNA on cell growth of the MCT4 negative cell line RT4 determined by MTT assay; Suppl. Fig 11. Effect of MCT4 silencing on cell growth in glucose free conditions determined by MTT assay; Suppl.Fig. 12. Effect of different concentrations of MCT4 specific antisense olignucleotide (ASO) on cell growth determined by MTT assay. Suppl. Fig. 13. Oxygen consumption rates at baseline (A), following glucose injection (B) and following oligomycin injection (C) in cell lines treated with control siRNA or MCT4 specific siRNAs.

Using sub-3-cycle pulses from mode-locked Cr:ZnS lasers at λ ≈ 2.4 µm as a driving source, we performed high-resolution dual-frequency-comb spectroscopy in the longwave infrared (LWIR) range. A duo of highly coherent broadband (6.6–11.4 µm) frequency combs were produced via intrapulse difference frequency generation in zinc germanium phosphide (ZGP) crystals. Fast (up to 0.1 s per spectrum) acquisition of 240,000 comb-mode-resolved data points, spaced by 80 MHz and referenced to a Rb clock, was demonstrated, resulting in metrology grade molecular spectra of N2O (nitrous oxide) and CH3OH (methane). The key to high-speed massive spectral data acquisition was low intensity and phase noise of the LWIR combs and high (7.5%) downconversion efficiency, resulting in a LWIR power of 300 mW for each comb.

Optical frequency combs based on mode-locked Cr:ZnS lasers uniquely combine multi-octave bandwidth, high brightness, and compactness. We demonstrate spectroscopy with dual Cr:ZnS combs accessing 800,000 comb teeth spanning 2000 cm–1 in the middle-IR.

We present recent results on high energy, high repetition rate 1645 nm Er:YAG laser optically pumped by efficient Er-doped CW fiber lasers. Up to 32.5 mJ pulse energy, 0.85 MW-level peak powers are obtained at 1000 Hz. The results are presented in a wide 500-5000 Hz range of repetition rates.

We report dual-comb spectroscopy with highly coherent broadband combs spanning 6– 12 µm produced via optical rectification of sub-three-cycle Cr:ZnS laser pulses. Real-time (~1sec) acquisition of broadband molecular spectra with the sub-Doppler resolution was demonstrated.

Focal therapy has emerged as a treatment option for low- to intermediate-risk localized prostate cancer (PCa) patients, to balance the risks for urinary and sexual morbidity of radical treatment with the psychological burden of active surveillance. In this context, we developed ST-4PC, an injectable, polymeric paste formulation containing docetaxel (dtx) and bicalutamide (bic) for image-guided focal therapy of PCa. The objective of this work was to evaluate the in vitro characteristics and in vivo toxicity and efficacy of ST-4PC. In vitro drug release was evaluated using high-performance liquid chromatography. In vivo toxicity of blank- and drug-loaded ST-4PC was assessed in mice and rats. Tumor growth inhibition was evaluated in LNCaP subcutaneous (s.c.) and LNCaP-luc orthotopic xenograft models. Using the s.c. model, mice were monitored weekly for weight loss, tumor volume (TV) and serum PSA. For the orthotopic model, mice were additionally monitored for bioluminescence as measure of tumor growth. ST-4PC demonstrated a sustained and steady release of incorporated drugs with 50% dtx and 20% bic being released after 14 days. While no systemic toxicity was observed, dose-dependent local side effects from dtx developed in the s.c. but not in the orthotopic model, illustrating the limitations of s.c. models for evaluating local cytotoxic therapy. In the s.c. model, 0.1%/4% and 0.25%/4% dtx/bic ST-4PC paste significantly reduced PSA progression, but did not have a significant inhibitory effect on TV. ST-4PC loaded with 1%/4% dtx/bic significantly reduced TV, serum PSA, and bioluminescence in the orthotopic xenograft model. Compared with drugs dissolved in DMSO, ST-4PC significantly delayed tumor growth. Image-guided focal therapy using ST-4PC demonstrated promising inhibition of PSA progression and orthotopic tumor growth in vivo without significant toxicity, and warrants further clinical evaluation.

Muscle-invasive bladder cancer represents approximately 25% of diagnosed bladder cancer cases and carries a significant risk of death. Oncolytic viruses are novel antitumor agents with the ability to selectively replicate and lyse tumor cells while sparing healthy tissue. We explored the efficiency of the oncolytic YB-1-selective adenovirus XVir-N-31 in vitro and in an orthotopic mouse model for bladder cancer by intramural injection under ultrasound guidance. We demonstrated that XVir-N-31 replicated in bladder cancer cells and induced a stronger immunogenic cell death than wild-type adenovirus by facilitating enhanced release of HMGB1 and exosomal Hsp70. The intratumoral delivery of XVir-N-31 by ultrasound guidance delayed tumor growth in an immunodeficient model, demonstrating the feasibility of this approach to deliver oncolytic viruses directly into the tumor.

A common form of treatment for patients with hepatocellular carcinoma (HCC) is transarterial radioembolization (TARE) with non-degradable glass or resin microspheres (MS) labeled with 90Y (90Y-MS). To further simplify the dosimetry calculations in the clinical setting, to have more control over the particle size and to change the permanent embolization to a temporary one, we developed uniformly-sized, biodegradable 188Re-labeled MS (188Re-MS) as a new and easily imageable TARE agent. Methods: MS made of poly(L-lactic acid) were produced in a flow focusing microchip. The MS were labeled with 188Re using a customized kit. An orthotopic HCC animal model was developed in male Sprague Dawley rats by injecting N1-S1 cells directly into the liver using ultrasound guidance. A suspension of 188Re-MS was administered via hepatic intra-arterial catheterization 2 weeks post-inoculation of the N1-S1 cells. The rats were imaged by SPECT 1, 24, 48, and 72 h post-radioembolization. Results: The spherical 188Re-MS had a diameter of 41.8 ± 6.0 µm (CV = 14.5%). The site and the depth of the injection of N1-S1 cells were controlled by visualization of the liver in sonograms. Single 0.5 g tumors were grown in all rats. 188Re-MS accumulated in the liver with no deposition in the lungs. 188Re decays to stable 188Os by emission of β¯ particles with similar energy to those emitted by 90Y while simultaneously emitting γ photons, which were imaged directly by single photon computed tomography (SPECT). Using Monte Carlo methods, the dose to the tumors was calculated to be 3-6 times larger than to the healthy liver tissue. Conclusions: 188Re-MS have the potential to become the next generation of β¯-emitting MS for TARE. Future work revolves around the investigation of the therapeutic potential of 188Re-MS in a large-scale, long-term preclinical study as well as the evaluation of the clinical outcomes of using 188Re-MS with different sizes, from 20 to 50 µm.

The significance of lactate transporters has been recognized in various cancer types, but their role in urothelial carcinoma remains mostly unknown. The aim of this study was to investigate the functional importance of the monocarboxylate transporter (MCT) 4 in preclinical models of urothelial carcinoma and to assess its relevance in patient tumors. The association of MCT4 expression with molecular subtypes and outcome was determined in The Cancer Genome Atlas (TCGA) cohort and two independent cohorts of patients with urothelial carcinoma. Silencing of MCT4 was performed using siRNAs in urothelial carcinoma cell lines. Effects of MCT4 inhibition on cell growth, apoptosis, and production of reactive oxygen species (ROS) were assessed. Moreover, effects on lactate efflux were determined. The in vivo effects of MCT4 silencing were assessed in an orthotopic xenograft model. MCT4 expression was higher in the basal subtype. Decreased MCT4 methylation and increased RNA and protein expression were associated with worse overall survival (OS). Inhibition of MCT4 led to a reduction in cell growth, induction of apoptosis, and an increased synthesis of ROS. MCT4 inhibition resulted in intracellular accumulation of lactate. In vivo, stable knockdown of MCT4 reduced tumor growth. The expression of MCT4 in urothelial carcinoma is associated with features of aggressive tumor biology and portends a poor prognosis. Inhibition of MCT4 results in decreased tumor growth in vitro and in vivo. Targeting lactate metabolism via MCT4 therefore provides a promising therapeutic approach for invasive urothelial carcinoma, especially in the basal subtype.

As an immune evasion and survival strategy, the Plasmodium falciparum malaria parasite has evolved a protein named VAR2CSA. This protein mediates sequestration of infected red blood cells in the placenta through the interaction with a unique carbohydrate abundantly and exclusively present in the placenta. Cancer cells were found to share the same expression of this distinct carbohydrate, termed oncofetal chondroitin sulfate on their surface. In this study we have used a protein conjugation system to produce a bispecific immune engager, V-aCD3, based on recombinant VAR2CSA as the cancer targeting moiety and an anti-CD3 single-chain variable fragment linked to a single-chain Fc as the immune engager. Conjugation of these two proteins resulted in a single functional moiety that induced immune mediated killing of a broad range of cancer cells in vitro and facilitated tumor arrest in an orthotopic bladder cancer xenograft model.

We demonstrate vector solitons in a few-cycle polycrystalline Cr:ZnS oscillator at the middle-IR wavelength 2.4 µm and deploy realistic simulations to identify χ(2) nonlinearity in a polycrystal as the main factor contributing to polarization dynamics.

We present recent results on 1645/1617 nm Er:YAG and 2090 nm Ho:YAG laser systems optically pumped by efficient CW fiber lasers. Multi-mJ pulse energy, MW-level peak powers are obtained in a range of repetition rates.

We report the first femtosecond polycrystalline Cr:ZnS oscillator with an octave-spanning spectrum. Average power of the laser is 1.4 W at the mid-IR central wavelength 2.4 μm and the pulse repetition frequency 79 MHz.

We report 45 dB single stage Cr:ZnSe amplifier operating over 2.2-2.6 μm spectral range seeded by CW Cr:ZnSe single frequency laser. The maximum output energy of 1.6 mJ was demonstrated in 25 ns pulses at 1000 Hz repetition rate.

We report dual-comb spectroscopy experiments with stabilized combs based on Kerr-lens mode-locked Cr:ZnS lasers. The compact dual-comb setup features >3W power per each channel, 80-MHz comb-mode spacing, and 60-THz-wide band centered at 2.4 μm.

We give an overview of experimental results on mid-IR femtosecond lasers and frequency combs based on polycrystalline Cr$^{2+}$: ZnS providing access to few-cycle pulses in the spectral range 2–$;3\mu \mathrm{m}$. We also discuss the techniques for generation of multi-octave coherent femtosecond continua with Cr$^{2+}$: ZnS lasers.

We report a technique for generation of ultra-broadband coherent femtosecond continua in the infrared. The laser architecture is based on the Cr:ZnS–GaSe and Cr:ZnS–ZGP tandem arrangements that enable simultaneous amplification of ultrashort middle IR pulses and augmentation of pulses’ spectrum via a chain of intrapulse three-wave mixings. The first part of the tandems is based on a single-pass polycrystalline Cr:ZnS amplifier, which is optically pumped by off-the-shelf continuous wave Er-doped fiber laser and outputs 2-cycle pulses with multi-Watt average power at 80 MHz repetition rate, at the central wavelength 2.5 μm. The second stage of the tandems comprises a GaSe or ZGP crystals configured for intrapulse difference frequency generation. The Cr:ZnS–GaSe tandem has allowed us to achieve multi-octave 2–20 μm continuum with 2 W power in the range 2–3 μm and power in excess of 20 mW in the important range 3–20 μm. On the other hand, Cr:ZnS–ZGP tandem features long-wave infrared (6–12 μm) output pulses with record braking sub-Watt power level. Last but not least, Cr:ZnS–GaSe and Cr:ZnS–ZGP IR sources have small footprints and are easily convertible to the optical frequency combs with low carrier-to-envelope timing jitter.

We report mid-IR frequency comb with 3.25 W average power and spectrum spanning 60 THz near 2.4 pm. We stabilized the offset frequency of the comb with accumulated phase error of 75 mrads.

We give an overview of recent experimental results on mid-IR femtosecond lasers and optical frequency combs based on TM:II-VI media providing access to few-cycle pulses with Watt-level power over 2–20 µm spectral range.

Enabling broad tunability, high peak and average power, ultrashort pulse duration, and all known modes of laser operation—transition-metal (TM)-doped II–VI chalcogenides are the materials of choice for direct lasing in the mid-IR. The host materials feature broad infrared transparency, high thermal conductivity, low phonon cutoff, low optical losses, and are available as either single crystals or polycrystalline ceramics. Doped with TM ions, these media exhibit a four-level energy structure, the absence of excited state absorption, as well as broad absorption and emission bands. Doped single-crystals of high optical quality are difficult to grow; however, the advent of postgrowth diffusion doped ceramics has resulted in significant progress in laser development. Here, we summarize recent experimental laser results on Cr and Fe doped II–VI chalcogenides providing access to the 1.8–6 μm spectral range with a high (>60%) efficiency, multi-Watt-level [140 W in continuous wave (CW)] output powers, tunability of >1000 nm, short-pulse (

We report, to the best of our knowledge, the first fully referenced Cr:ZnS optical frequency comb. The comb features few cycle output pulses with 3.25 W average power at 80 MHz repetition rate, spectrum spanning 60 THz in the middle-IR range 1.79–2.86 µm, and a small footprint (0.1 m2), The spectral components used for the measurement of the comb’s carrier envelope offset frequency were obtained directly inside the polycrystalline Cr:ZnS laser medium via intrinsic nonlinear interferometry. Using this scheme we stabilized the offset frequency of the comb with the residual phase noise of 75 mrads.

Objective Carbonic anhydrase IX (CA9) is important in the regulation of intra- and extracellular pH in solid tumors, contributing to cell growth and invasion. In urothelial carcinoma (UC), CA9 has been identified as a urinary marker for disease detection, but its biologic role is unknown. To date, differential gene expression patterns of CA9 in various molecular subtypes and potential effects of CA9 inhibition in UC cells are unknown. We aimed to investigate the function of CA9 and the effects of CA9 inhibition in invasive UC. Methods Immunohistochemistry was used to assess CA9 expression in a cohort of 153 patients undergoing radical cystectomy. CA9 expression was correlated with molecular subtype by analysis of the TCGA data and of our own cohort of 223 patients with invasive UC receiving neoadjuvant chemotherapy. CA9 expression was assessed in a panel of 12 UC cell lines by Western Blot and qPCR, and multiple siRNAs were used to silence CA9 in 2 cell lines. Effects of CA9 silencing on cell growth, migration, and invasion were assessed. We also used the small molecule inhibitor U-104 to inhibit CA9 in vitro and in an orthotopic xenograft model. Results CA9 expression was higher in cancer tissue compared to benign urothelium and was particularly highly expressed in luminal papillary and basal squamous tumors. CA9 expression did not correlate with outcome after neoadjuvant chemotherapy and/or radical cystectomy. Silencing of CA9 by siRNA diminished invasion but did not induce a consistent change of cell growth and migration. Treatment with U-104 led to cell growth reduction only at high concentrations in vitro and failed to have a significant effect on tumor growth in vivo. Conclusions The present study confirms over-expression of CA9 in UC and for the first time shows a correlation with molecular subtypes. However, CA9 expression showed no association with the outcome of patients with muscle invasive bladder cancer and inhibition of CA9 did not lead to a consistent inhibition of tumor growth. Based on these data, CA9 exhibits a role neither as a predictive or prognostic marker nor as a therapeutic target in invasive UC.

Background The vast majority of prostate cancer presents clinically localized to the prostate without evidence of metastasis. Currently, there are several modalities available to treat this particular disease. Despite radical prostatectomy demonstrating a modest prostate cancer specific mortality benefit in the PIVOT trial, several novel modalities have emerged to treat localized prostate cancer in patients that are either not eligible for surgery or that prefer an alternative approach. Methods Athymic nude mice were subcutaneously inoculated with prostate cancer cells. The mice were divided into four cohorts, one cohort untreated, two cohorts received docetaxel (10 mg/kg) either subcutaneously (SC) or intravenously (IV) and the fourth cohort was treated using the magnetically-actuated docetaxel delivery device (MADDD), dispensing 1.5 μg of docetaxel per 30 min treatment session. Treatment in all three therapeutic arms (SC, IV, and MADDD) was administered once weekly for 6 weeks. Treatment efficacy was measured once a week according to tumor volume using ultrasound. In addition, calipers were used to assess tumor volume. Results Animals implanted with the device demonstrated no signs of distress or discomfort, neither local nor systemic symptoms of inflammation and infection. Using an independent sample t-test, the tumor growth rate of the treated tumors was significant when compared to the control. Post hoc Tukey HSD test results showed that the mean tumor growth rate of our device cohort was significantly lower than SC and control cohorts. Moreover, IV cohort showed slight reduction in mean tumor growth rates than the ones from the device cohort, however, there was no statistical significance in tumor growth rate between these two cohorts. Furthermore, immunohistochemistry demonstrated an increased cellular apoptosis in the MADDD treated tumors and a decreased proliferation when compared to the other cohorts. In addition, IV cohort showed increased treatment side effects (weight loss) when compared to the device cohort. Finally, MADDD showed minimal expression of CD45 comparable to the control cohort, suggesting no signs of chronic inflammation. Conclusions In conclusion, this study showed for the first time that MADDD, clearly suppressed tumor growth in local prostate cancer tumors. This could potentially be a novel clinical treatment approach for localized prostate cancer.

The acquisition of an invasive phenotype by epithelial cells occurs through a loss of cellular adhesion and polarity, heralding a multistep process that leads to metastatic dissemination. Since its characterization in 1995, epithelial-mesenchymal transition (EMT) has been closely linked to the metastatic process. As a defining aspect of EMT, loss of cell adhesion through downregulation of E-cadherin is carried out by several transcriptional repressors; key among them the SNAI family of transcription factors. Here we identify for the first time that Lyn kinase functions as a key modulator of SNAI family protein localization and stability through control of the Vav-Rac1-PAK1 (Vav-Rac1-p21-activated kinase) pathway. Accordingly, targeting Lyn in vitro reduces EMT and in vivo reduces metastasis of primary tumors. We also demonstrate the clinical relevance of targeting Lyn as a key player controlling EMT; patient samples across many cancers revealed a strong negative correlation between Lyn and E-cadherin, and high Lyn expression in metastatic tumors as well as metastasis-prone primary tumors. This work reveals a novel pancancer mechanism of Lyn-dependent control of EMT and further underscores the role of this kinase in tumor progression.

We demonstrate a fs laser architecture that enables the direct measurement of the laser’s carrier envelope offset frequency. A multi-Watt source is arranged as a full-repetition-rate polycrystalline Cr:ZnS MOPA and covers 3-octaves (0.4–4.2 µm).

Until recently, the generation of super-octave continua in bulk materials at full repetition rates of femtosecond (fs) oscillators has been limited to a few special cases of Kerr lens mode-locked Ti:sapphire lasers. In 2019, we described a 3.4-octave fs source with 50 nJ pulse energy at the repetition rate of 78 MHz based on polycrystalline Cr:ZnS [Vasilyev et al., Optica 6, 126 (2019)OPTIC82334-253610.1364/OPTICA.6.000126]. Here we explain the mechanism of fs supercontinuum generation in transition-metal doped polycrystalline II-VI semiconductors at relatively low (nJ-level) pulse energy. We demonstrate that this new supercontinuum regime is enabled by a complex, yet well-reproducible, interplay between the effects arising from the third-order nonlinearity, the quadratic nonlinearities, and thermal optical effects in the medium. We also demonstrate the control of fs pulse propagation in disordered χ ( 2 ) media via the control of the material microstructure.

Background Intravesical instillation of chemo- or immunotherapy is commonly used in bladder cancer. Upper tract urothelial carcinoma (UTUC) shares similar pathological features, but current formulations are not suitable for direct instillation to the upper urinary tract. Objective To evaluate in vivo applicability, characteristics and toxicity of ST-UC, a mucoadhesive polymeric paste formulation of gemcitabine, for upper urinary tract instillation. Material and methods Three pigs received 10 ml of ST-UC (100 mg/ml gemcitabine) retrogradely into 1 renal pelvis for pharmacokinetic studies. Four days later, a second injection into the contralateral renal pelvis was followed by serial euthanasia of the pigs and nephroureterectomy after 1, 3, and 6 hours. Adverse effects were monitored. Urine, serum, and tissue gemcitabine concentrations were measured, along with histologic examination of the upper urinary tract. Results Retrograde instillation of ST-UC was well tolerated with mild, completely receding hydronephrosis. Urine gemcitabine concentrations were highest in the first 3-hour collection interval. Hundred percent of gemcitabine was recovered in the urine within 24 hours. Serum peak concentrations (cmax) of gemcitabine were low at 5.5 µg/ml compared to the 10 to 30 µg/ml levels observed after a single intravenous dose of 1,000 mg/m2 gemcitabine. The formulation was still traceable after one hour and gemcitabine tissue concentrations are supportive of this extended drug exposure. No major histopathological changes were observed. The main limitation of this study is the lack of antitumor activity data. Conclusion This preclinical evaluation of ST-UC demonstrated feasible instillation in the renal pelvis, no significant safety concerns, and sustained release of gemcitabine.

We report, to the best of our knowledge, the first super-octave femtosecond polycrystalline Cr:ZnS laser at the central wavelength 2.4 µm. The laser is based on a non-polarizing astigmatic X-folded resonator with normal incidence mounting of the gain element. The chromatic dispersion of the resonator is controlled with a set of dispersive mirrors within one third of an optical octave over 2.05–2.6 µm range. The resonator’s optics is highly reflective in the range 1.8–2.9 µm. The components of the oscillator’s output spectrum at the wavelengths 1.6 µm and 3.2 µm are detected at –60 dB with respect to the main peak. Average power of few-cycle Kerr-lens mode-locked laser is 1.4 W at the pulse repetition frequency 79 MHz. That corresponds to 22% conversion of cw radiation of Er-doped fiber laser, which we used for optical pumping of the Cr:ZnS oscillator.

Introduction Bladder cancer is among the top ten most common cancer types in the world, with approximately 550,000 new cases annually. The highest burden of bladder cancer is currently on most developed communities across the world and an estimated 9,000 Canadians are diagnosed with bladder cancer each year. Cisplatin-based Neoadjuvant chemotherapy (NAC) followed by radical cystectomy in patients with muscle-invasive bladder cancer (MIBC) has been shown to improve five-year survival, and is therefore currently the first-line standard of care in patients. However, 60% of patients are inherently resistant to NAC at the time of cystectomy. While several mechanisms of cellular resistance to cisplatin have been proposed, the mechanisms presented thus far still do not offer and effective patient response prediction in the context of MIBC. There is therefore, an urgent and unmet need to determine clinically actionable mechanisms of cisplatin resistance. Methods In order to elucidate mechanisms of resistant to cisplatin, the study presented in this thesis takes advantage of a pooled genome‐wide CRISPR knock‐out library targeting 19,114 protein coding genes with 76,441 synthetic guide RNAs (sgRNAs) which allows for an unbiased screen. Upon completion of the screen the top hit was validated in vitro (CRISPR knockout cell lines), in vivo (mouse models) and in patient tumour tissue samples by immunohistochemistry. Results A full-scale screen revealed that several genes involved in the pro-apoptotic pathway (such as CASP8, BAX, and TNFSFR10A) and cell cycle regulation have the potential to confer resistance to cisplatin when knocked out. For this study however, we validated the top hit from our screen - Schlafen 11 (SLFN11) - and established that the complete loss of SLFN11 confers a cisplatin resistant phenotype in MIBC. We further established that SLFN11 is involved in the regulation of cell cycle progression upon cisplatin challenge and does so via interactions with Mediator of DNA Damage Checkpoint 1 (MDC1) protein. Conclusions Overall, the study presented here offers SLFN11 as a potential biomarker to aid in clinical decision making and to anticipate resistance to cisplatin-based NAC in MIBC. Furthermore, targeting SLFN11 associated pathways could allow for the development of combination therapies to be used in conjunction with cisplatin in the future.

We have recently demonstrated a critical role for progranulin in bladder cancer. Progranulin contributes, as an autocrine growth factor, to the transformed phenotype by modulating Akt-and MAPK-driven motility, invasion and anchorage-independent growth. Progranulin also induces F-actin remodeling by interacting with the F-actin binding protein drebrin. In addition, progranulin is overexpressed in invasive bladder cancer compared to normal tissue controls, suggesting that progranulin might play a key role in driving the transition to the invasive phenotype of urothelial cancer. However, it is not established whether targeting progranulin could have therapeutic effects on bladder cancer. In this study, we stably depleted urothelial cancer cells of endogenous progranulin by shRNA approaches and determined that progranulin depletion severely inhibited the ability of tumorigenic urothelial cancer cells to migrate, invade and grow in anchorage-independency. We further demonstrate that progranulin expression is critical for tumor growth in vivo, in both xenograft and orthotopic tumor models. Notably, progranulin levels correlated with response to cisplatin treatment and were upregulated in bladder tumors. Our data indicate that progranulin may constitute a novel target for therapeutic intervention in bladder tumors. In addition, progranulin may serve as a novel biomarker for bladder cancer.

We have developed a murine intravesical orthotopic human bladder cancer (mio-hBC) model for the establishment of superficial urothelial cell carcinomas. In this model we catheterize female atyhmic nude mice and pre-treat the bladder with poly-L-lysine for 15 minutes, followed by intravesical instillation of luciferase-transfected human UM-UC-3 cells. Cancer cells are quantified by bioluminescent imaging which has been validated by small animal ultrasound. Poly-L-lysine pre-treatment increased engraftment rate (84.4%) and resulted in faster growing tumors than trypsin pre-treatment. In addition, tumors respond through a decrease in growth and increase in apoptosis to chemotherapy with mitomycin C. Previous intravesical models utilized KU7 cells which have been later determined to be of non-bladder origin. They display markers consistent with HeLa cells, requiring a need for a true intravesical bladder model. Efficient engraftment and rapid superficial growth patterning of the human bladder tumor differentiate this in vivo orthotopic model from previous bladder models.

The sonic hedgehog (SHH) signaling pathway plays an integral role in the maintenance and progression of bladder cancer (BCa) and SHH inhibition may be an efficacious strategy for BCa treatment. We assessed an in-house human BCa tissue microarray and found that the SHH transcription factors, GLI1 and GLI2, were increased in disease progression. A panel of BCa cell lines show that two invasive lines, UM-UC-3 and 253J-BV, both express these transcription factors but UM-UC-3 produces more SHH ligand and is less responsive in viability to pathway stimulation by recombinant human SHH or smoothened agonist, and less responsive to inhibitors including the smoothened inhibitors cyclopamine and SANT-1. In contrast, 253J-BV was highly responsive to these manipulations. We utilized a GLI1 and GLI2 antisense oligonucleotide (ASO) to bypass pathway mechanics and target the transcription factors directly. UM-UC-3 decreased in viability due to both ASOs but 253J-BV was only affected by GLI2 ASO. We utilized the murine intravesical orthotopic human BCa (mio-hBC) model for the establishment of noninvasive BCa and treated tumors with GLI2 ASO. Tumor size, growth rate, and GLI2 messenger RNA and protein expression were decreased. These results suggest that GLI2 ASO may be a promising new targeted therapy for BCa.

II-VI binary and ternary chalcogenides (e.g. ZnS, ZnSe; CdZnTe, ) doped with transition metal (TM) ions such as Cr, and Fe are arguably the materials of choice for effective mid-IR lasers potentially covering 1.8-9 µm spectral range. This talk summarizes progress in Cr:ZnS/Se and Fe:ZnSe laser systems, enabling a wide range of tunability (1.8-5.0µm) with output power levels of up to 140 W, as well as Fe doped ternary chalcogenides with tunability potentially extended up to 9 um. TM:II-VI media feature a unique combination of superb ultra-fast laser capabilities with high nonlinearity enabling exceptional output characteristics of polycrystalline Cr:ZnS/Se oscillators in Kerr-Lens-Mode-Locked (KLM) regime over 2-2.6 um and effective up and down conversion of fs pulses via random phase matching (RFM). Extension of mid-IR spectral coverage to 3-8 um is demonstrated by Cr:ZnS KLM laser pumped subharmonic parametric oscillators (OPOs) based on quasi-phase matching in OP-GaAs, and RFM in polycrystalline ZnSe. Fe:II-VI semiconductors are complimentary to Cr doped compounds and 3-8 um KLM ultrafast oscillators based on Fe doped chalcogenides are feasible. Another important feature of Fe:II-VI media is excellent energy storage capability at 77-200K (~60 µs luminescence life time) enabling efficient Q-switched regime and high energy amplification of ns and ultrafast pulses. One of the major problems in the development of CW, gain switched, Q-switched and KLM ultrafast Fe:II-VI lasers was the absence of convenient pump sources overlapping with absorption band (2.7-4.5 um) of Fe: gain media. Potential utilization of Quantum Cascade Lasers (QCL) as pump sources of Fe:II-VI lasers will be discussed in the form QCL-solid state laser hybrid platforms as well as Fe doped active layers integrated in QCL structures.

II-VI chalcogenides (e.g. ZnSe/S) doped with transition metal (TM) ions such as Cr, and Fe are arguably the materials of choice for fabrication of effective mid-IR gain media. TM:II-VI materials feature a favorable blend of laser spectroscopic parameters: a four-level energy structure, absence of excited state absorption, close to 100% quantum efficiency of fluorescence (for Cr doped II-VI media), broad mid-IR vibronic absorption and emission bands. This talk summarizes progress in fabrication of high quality Cr:ZnS/Se and Fe:ZnS/Se by cation vacancy and cation interstitial enhanced post growth thermal diffusion. We also describe recent breakthrough on recrystallization and effective doping of ZnS ceramics under hot isostatic pressing resulting in a large cm-scale monocrystalline domains formation and an increase of the Fe diffusion coefficient by three orders of magnitude. We report recent advances in high-power Cr:ZnS/Se and Fe:ZnSe laser systems, enabling a wide range of tunability (1.8-5.0µm) with output power levels of up to 140 W near 2500 nm, 32 W at 2940 nm, and 35 W at 4300 nm with corresponding optical efficiencies of 62%, 29%, and 35%. Current improvements of output characteristics of polycrystalline Cr:ZnS/Se oscillators in Kerr-Lens-Mode-Locked (KLM) regime are reported: up to 2 W output power at 75-1200 MHz repetition rate, up to 2 cycle pulse duration (16 fs) with efficiency of 20-25% with regards to Er-fiber laser pump power. The effects of efficient up-conversion of mid-IR fs pulses in the laser medium as well as supercontinuum generation are demonstrated. Further extension of mid-IR spectral coverage to 3-8 m is demonstrated by Cr:ZnS KLM laser pumped degenerate (subharmonic) parametric oscillators (OPOs) based on based on quasi-phase matching in orientation-patterned gallium arsenide, and random phase matching in polycrystalline ZnSe.

Frequency combs with a wide instantaneous spectral span covering the 3–20 µm molecular fingerprint region are highly desirable for broadband and high-resolution frequency comb spectroscopy, trace molecular detection, and remote sensing. We demonstrate a novel approach for generating high-average-power middle-infrared (MIR) output suitable for producing frequency combs with an instantaneous spectral coverage close to 1.5 octaves. Our method is based on utilizing a highly-efficient and compact Kerr-lens mode-locked Cr2+:ZnS laser operating at 2.35-µm central wavelength with 6-W average power, 77-fs pulse duration, and high 0.9-GHz repetition rate; to pump a degenerate (subharmonic) optical parametric oscillator (OPO) based on a quasi-phase-matched GaAs crystal. Such subharmonic OPO is a nearly ideal frequency converter capable of extending the benefits of frequency combs based on well-established mode-locked pump lasers to the MIR region through rigorous, phase- and frequency-locked down conversion. We report a 0.5-W output in the form of an ultra-broadband spectrum spanning 3–8 µm measured at 50-dB level.

We report compact ultrafast mid-IR sources based on single-pass cw and pulsed pumped polycrystalline Cr:ZnS/Se laser amplifiers exhibiting up to 37% efficiency, up to 30 dB small-signal gain, and spectral span of 1.6–4.5 um.

We demonstrate an approach to power scaling of middle-IR femtosecond pulses at the wavelength 2-3 μm to multi ten-Watt level in simple and robust single-pass spinning ring amplifier based on polycrystalline Cr:ZnSe.