Live vaccines are ideal for inducing immunity but suffer from the need to attenuate their pathogenicity or replication to preclude the possibility of escape. Unnatural amino acids (UAAs) provide a strategy to engineer stringent auxotrophies, yielding conditionally replication incompetent live bacteria with excellent safety profiles. Here, we engineer Pseudomonas aeruginosa to maintain auxotrophy for the UAA p-benzoyl-L-phenylalanine (BzF) through its incorporation into the essential protein DnaN. In vivo evolution using an Escherichia coli-based two-hybrid selection system enabled engineering of a mutant DnaN homodimeric interface completely dependent on a BzF-specific interaction. This engineered strain, Pa Vaccine, exhibits undetectable escape frequency (<10-11) and shows excellent safety in naïve mice. Animals vaccinated via intranasal or intraperitoneal routes are protected from lethal challenge with pathogenic P. aeruginosa PA14. These results establish UAA-auxotrophic bacteria as promising candidates for bacterial vaccine therapy and outline a platform for expanding this technology to diverse bacterial pathogens.

Platinum-resistant human epithelial ovarian cancer cell lines OVCAR-5 and OVCAR-4 expresses tyrosyl-DNA phosphodiesterase 1 (Tdp1). The Tdp1 protein expression levels in human epithelial ovarian carcinoma cell lines OVCAR-5 (derived from the ascitic fluid of EOC patient without prior treatment; tumor characterized to be platinum-resistant) and OVCAR-4 (derived from the ascitic fluid of platinum-refractory EOC patient) were evaluated by Western Blot. Tdp1 expression was found higher in the OVCAR-4 cells compared to that in the OVCAR-5 cells.

Preliminary analysis of costs related to the side effect management for irinotecan-based combination therapies.

Supplementary Methods Supplemental Figure 1: The MIA-PaCa-2 + PCAF model exhibits histologic features consistent with desmoplasia 90 days post implantation Supplemental Figure 2: 1 μM calcipitriol activates the vitamin D receptor (VDR) in cancer associated fibroblasts Supplemental Figure 3: VDAR activation + PDP enable a 75% dose reduction in nal-IRI Supplemental Figure 4: Inhibition of the CXCL12/CXCR7 by CAL+PDP is lost by day 90 post-implantation Supplemental Figure 5: Calcipotriol and photodynamic priming cytotoxicity curves Supplemental Figure 6: MRC-5 conditioned medium does not affect the baseline metabolic activity of PDAC cell lines Supplemental Figure 7: PDP increases intratumoral nal-IRI via increased vascular and parenchymal permeability Supplemental Figure 8: Treatment with CAL+PDP+5 mg/kg nal-IRI induces global transcriptomic shifts in pancreataic tumors that correlate with a less aggressive and more responsive treatment profile compared to treatment with PDP+20 mg/kg nal-IRI

Comparison of minocycline and irinotecan doses used in this study versus in the clinic.

Preliminary analysis of combining minocycline and irinotecan regimen indicates significant cost savings compared to other irinotecan-based combination therapies.

Physical characterizations of nanoliposomal irinotecan (nal-IRI) for in vivo study.

Representative size distribution of nanoliposomal irinotecan (nal-IRI) and absorbance spectrum of nal-IRI in DMSO. (A) Mono-dispersed nal-IRI is approximately 115 nm in diameter with a Polydispersity index below 0.1. (B) Nal-IRI absorbance spectrum was measured in DMSO, and the irinotecan concentration was determined by UV-Vis spectroscopy with appropriate standard curves.

Numerous cancer models have been developed to investigate the effects of mechanical stress on the biology of cells. Here we describe a protocol to fabricate a perfusion model to culture 3-dimensional (3D) ovarian cancer nodules under constant flow. The modular design of this model allows for a wide range of treatment regimens and combinations, including PDT and chemotherapy. Finally, methods for a number of readouts are detailed, allowing researchers to investigate a variety of biological and cytotoxic parameters related to mechanical stress and therapeutic modalities.

Despite substantial drug development efforts, pancreatic adenocarcinoma (PDAC) remains a difficult disease to treat, and surgical resection is the only potentially curative option. Unfortunately, 80% of patients are ineligible for surgery due to the presence of invasive disease and/or distant metastases at the time of diagnosis. Treatment strategies geared towards reclassifying these patients as surgical candidates by reducing metastatic burden represents the most promising approach to improve long-term survival. We describe a photodynamic therapy (PDT) based approach that, in combination with the first-line chemotherapeutic nab-paclitaxel, effectively addresses distant metastases in three separate orthotopic PDAC models in immunodeficient mice. In addition to effectively controlling local tumor growth, PDT plus nab-paclitaxel primes the tumor to elicit systemic effects and reduce or abrogate metastases. This combination dramatically inhibits (up to 100%) the eventual development of metastases in models of early stage PDAC, and completely eliminates metastasis in 55% of animals with already established distant disease in late-stage models. Our findings suggest that this light activation process initiates local biological and/or physiological changes within the tumor microenvironment that can be leveraged to treat both localized and distant disease, and potentially reclassify patients with previously inoperable disease as surgical candidates.

Abstract Pancreatic cancer is a devastating disease for which the 5 year survival rate remains < 10% despite years of concerted research efforts. Potentially curative surgical resection cannot even be offered to most patients due to the presence of distant metastases at the time of diagnosis. Here, we present a neoadjuvant strategy comprised of photochemistry mediated tumor modulation combined with systemic therapy to significantly cytoreduce, and in 50% of cases completely eliminate, distant metastases to create a window of potential resectability. In a late stage model of pancreas cancer in which distant metastases are present at the time of treatment, combined photodynamic therapy (PDT) and gemcitabine/abraxane led to a 10,000 fold decrease in the number of distant metastases compared to untreated controls. In comparison, gemcitabine + abraxane, the current standard of care for pancreas cancer, only led to a 100 fold cytoreduction. 50% of animals treated with PDT + gem/abraxane harbored no detectable metastases 51 days after treatment compared to 0% of gem/abraxane treated animals, suggesting that PDT plus cytoreduction could enable potentially curative surgery in a sizeable percentage of animals. The strategy presented here is rapidly translatable and holds significant promise to dramatically increase the percentage of patients that are currently eligible for curative therapy for this devastating disease. Citation Format: Sriram Anbil, Zhiming Mai, Michael Pigula, Tayyaba Hasan. Photochemistry mediated tumor priming potentiates cytoreduction of distant metastases to enable potential surgical cure in a late stage model of pancreas cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-052.

Abstract Patients with cancer often confront the decision of whether to continue high-dose chemotherapy at the expense of cumulative toxicities. Reducing the dose of chemotherapy regimens while preserving efficacy is sorely needed to preserve the performance status of these vulnerable patients, yet has not been prioritized. Here, we introduce a dual pronged approach to modulate the microenvironment of desmoplastic pancreatic tumors and enable significant dose deescalation of the FDA-approved chemotherapeutic nanoliposomal irinotecan (nal-IRI) without compromising tumor control. We demonstrate that light-based photodynamic priming (PDP) coupled with vitamin D3 receptor (VDR) activation within fibroblasts increases intratumoral nal-IRI accumulation and suppresses protumorigenic CXCL12/CXCR7 crosstalk. Combined photodynamic and biochemical modulation of the tumor microenvironment enables a 75% dose reduction of nal-IRI while maintaining treatment efficacy, resulting in improved tolerability. Modifying the disease landscape to increase the susceptibility of cancer, via preferentially modulating fibroblasts, represents a promising and relatively underexplored strategy to enable dose deescalation. The approach presented here, using a combination of three clinically available therapies with nonoverlapping toxicities, can be rapidly translated with minimal modification to treatment workflow, and challenges the notion that significant improvements in chemotherapy efficacy can only be achieved at the expense of increased toxicity.

A key reason for the persistently grim statistics associated with metastatic ovarian cancer is resistance to conventional agents, including platinum-based chemotherapies. A major source of treatment failure is the high degree of genetic and molecular heterogeneity, which results from significant underlying genomic instability, as well as stromal and physical cues in the microenvironment. Ovarian cancer commonly disseminates via transcoelomic routes to distant sites, which is associated with the frequent production of malignant ascites, as well as the poorest prognosis. In addition to providing a cell and protein-rich environment for cancer growth and progression, ascitic fluid also confers physical stress on tumors. An understudied area in ovarian cancer research is the impact of fluid shear stress on treatment failure. Here, we investigate the effect of fluid shear stress on response to platinum-based chemotherapy and the modulation of molecular pathways associated with aggressive disease in a perfusion model for adherent 3D ovarian cancer nodules. Resistance to carboplatin is observed under flow with a concomitant increase in the expression and activation of the epidermal growth factor receptor (EGFR) as well as downstream signaling members mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and extracellular signal-regulated kinase (ERK). The uptake of platinum by the 3D ovarian cancer nodules was significantly higher in flow cultures compared to static cultures. A downregulation of phospho-focal adhesion kinase (p-FAK), vinculin, and phospho-paxillin was observed following carboplatin treatment in both flow and static cultures. Interestingly, low-dose anti-EGFR photoimmunotherapy (PIT), a targeted photochemical modality, was found to be equally effective in ovarian tumors grown under flow and static conditions. These findings highlight the need to further develop PIT-based combinations that target the EGFR, and sensitize ovarian cancers to chemotherapy in the context of flow-induced shear stress.

We developed a carbapenemase test based on the ability of imipenem to inhibit noncarbapenemase β-lactamases. The test uses bacterial isolates with a fluorescent β-lactamase substrate, producing objective results with 100% sensitivity and specificity in 10 minutes. The assay is inexpensive and consists of only 1 mixing step.

AbstractBackgroundPhotoimmunotherapy involves targeted delivery of photosensitizers via an antibody conjugate (i.e., photoimmunoconjugate, PIC) followed by light activation for selective tumor killing. The trade-off between PIC selectivity and PIC uptake is a major drawback limiting the efficacy of photoimmunotherapy. Despite ample evidence showing that photoimmunotherapy is most effective when combined with chemotherapy, the design of nanocarriers to co-deliver PICs and chemotherapy drugs remains an unmet need. To overcome these challenges, we developed a novel photoimmunoconjugate-nanoliposome (PIC-Nal) comprising of three clinically used agents: anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody cetuximab (Cet), benzoporphyrin derivative (BPD) photosensitizer, and irinotecan (IRI) chemotherapy.ResultsThe BPD photosensitizers were first tethered to Cet at a molar ratio of 6:1 using carbodiimide chemistry to form PICs. Conjugation of PICs onto nanoliposome irinotecan (Nal–IRI) was facilitated by copper-free click chemistry, which resulted in monodispersed PIC–Nal–IRI with an average size of 158.8 ± 15.6 nm. PIC–Nal–IRI is highly selective against EGFR-overexpressing epithelial ovarian cancer cells with 2- to 6-fold less accumulation in low EGFR expressing cells. Successful coupling of PIC onto Nal–IRI enhanced PIC uptake and photoimmunotherapy efficacy by up to 30% in OVCAR-5 cells. Furthermore, PIC–Nal–IRI synergistically reduced cancer viability via a unique three-way mechanism (i.e., EGFR downregulation, mitochondrial depolarization, and DNA damage).ConclusionIt is increasingly evident that the most effective therapies for cancer will involve combination treatments that target multiple non-overlapping pathways while minimizing side effects. Nanotechnology combined with photochemistry provides a unique opportunity to simultaneously deliver and activate multiple drugs that target all major regions of a cancer cell—plasma membrane, cytoplasm, and nucleus. PIC–Nal–IRI offers a promising strategy to overcome the selectivity-uptake trade-off, improve photoimmunotherapy efficacy, and enable multi-tier cancer targeting. Controllable drug compartmentalization, easy surface modification, and high clinical relevance collectively make PIC–Nal–IRI extremely valuable and merits further investigations in living animals.

A range of cellular, architectural, and physical cues in the tumor microenvironment influence the intrinsic and acquired resistance mechanisms that lead to treatment failure. Strategies that leverage photodynamic therapy (PDT), a photochemistry-based biophysical treatment modality, to regionally target and prime stubborn tumor populations may be essential to realizing durable improvements in cancer management while minimizing toxicity from traditional agents. Capturing these attributes in rationally-designed combinations has shown promise by synergistically reducing tumor area in 3D models, and durably controlling tumor burden in vivo. Among the areas that remain understudied is the influence of mechanical forces, such as hydrodynamic shear stress, on resistance, and the development of 3D tumor models and in vivo models that account for physical stress. To evaluate and optimize PDT regimens, and PDT-based combinations, designed to overcome resistance to conventional therapies due to physical stress, a multi-faceted approach is needed. Here the impact of hydrodynamic stress is evaluated in bioengineered 3D tumor models in the context of ovarian cancer. The potential value of using biologically inspired in vitro models to guide customized, rationally-designed PDT-based combination regimens will be presented.

Cancer patients often must confront the decision of whether to continue high dose chemotherapy at the expense of cumulative toxicities and high cost. Strategies to reduce the toxicity of these high dose regimens, while also retaining efficacy, have not been prioritized yet are sorely needed to preserve the performance status of these vulnerable patients. Here, we introduce a dual pronged approach to modulate the microenvironment of desmoplastic pancreatic tumors and enable significant dose de-escalation of the FDA approved frontline chemotherapeutic nanoliposomal irinotecan (nal-IRI) without compromising long-term tumor control. We demonstrate that light-based photodynamic priming (PDP) coupled with vitamin D receptor (VDR) activation targets cancer-associated microvasculature and fibroblasts to increase intratumoral nal-IRI and suppress pro-tumorigenic CXCL12/CXCR7 crosstalk. Combined optical and biochemical alteration of the tumor microenvironment enhances the efficacy of nal-IRI to enable a 75% dose de-escalation, resulting in improved tolerability with retained efficacy. Strategies aimed at modifying the tumor landscape to increase susceptibility represents a promising and relatively underexplored approach to enable dose de-escalation of toxic chemotherapeutics, and may simultaneously improve patient outcomes and quality-of-life.

Ovarian cancer (OvCa) is the leading cause of gynecological cancer-related deaths in the United States, with five-year survival rates of 15-20% for stage III cancers and 5% for stage IV cancers. The standard of care for advanced OvCa involves surgical debulking of disseminated disease in the peritoneum followed by chemotherapy. Despite advances in treatment efficacy, the prognosis for advanced stage OvCa patients remains poor and the emergence of chemoresistant disease localized to the peritoneum is the primary cause of death. Therefore, a complementary modality that is agnostic to typical chemo- and radio-resistance mechanisms is urgently needed. Photodynamic therapy (PDT), a photochemistry-based process, is an ideal complement to standard treatments for residual disease. The confinement of the disease in the peritoneal cavity makes it amenable for regionally localized treatment with PDT. PDT involves photochemical generation of cytotoxic reactive molecular species (RMS) by non-toxic photosensitizers (PSs) following exposure to non-harmful visible light, leading to localized cell death. However, due to the complex topology of sensitive organs in the peritoneum, diffuse intra-abdominal PDT induces dose-limiting toxicities due to non-selective accumulation of PSs in both healthy and diseased tissue. In an effort to achieve selective damage to tumorous nodules, targeted PS formulations have shown promise to make PDT a feasible treatment modality in this setting. This targeted strategy involves chemical conjugation of PSs to antibodies, referred to as photoimmunoconjugates (PICs), to target OvCa specific molecular markers leading to enhanced therapeutic outcomes while reducing off-target toxicity. In light of promising results of pilot clinical studies and recent preclinical advances, this review provides the rationale and methodologies for PIC-based PDT, or photo-immunotherapy (PIT), in the context of OvCa management.

Abstract Mechanism-inspired drug repurposing that augments standard treatments offers a cost-effective and rapid route toward addressing the burgeoning problem of plateauing of effective therapeutics for drug-resistant micrometastases. We show that the antibiotic minocycline, by its ability to minimize DNA repair via reduced expression of tyrosyl-DNA phosphodiesterase-1 (Tdp1), removes a key process attenuating the efficacy of irinotecan, a frequently used chemotherapeutic against metastatic disease. Moreover, minocycline and irinotecan cooperatively mitigate each other's undesired cytokine inductions of VEGF and IL8, respectively, thereby reinforcing the benefits of each modality. These mechanistic interactions result in synergistic enhancement of irinotecan-induced platinum-resistant epithelial ovarian cancer cell death, reduced micrometastases in the omenta and mesentery by >75%, and an extended overall survival by 50% in a late-stage peritoneal carcinomatosis mouse model. Economic incentives and easy translatability make the repurposing of minocycline as a reinforcer of the topoisomerase class of chemotherapeutics extremely valuable and merits further investigations. Mol Cancer Ther; 17(2); 508–20. ©2017 AACR.

The past three decades have witnessed notable advances in establishing photosensitizer-antibody photo-immunoconjugates for photo-immunotherapy and imaging of tumors. Photo-immunotherapy minimizes damage to surrounding healthy tissue when using a cancer-selective photo-immunoconjugate, but requires a threshold intracellular photosensitizer concentration to be effective. Delivery of immunoconjugates to the target cells is often hindered by I) the low photosensitizer-to-antibody ratio of photo-immunoconjugates and II) the limited amount of target molecule presented on the cell surface. Here, a nanoengineering approach is introduced to overcome these obstacles and improve the effectiveness of photo-immunotherapy and imaging. Click chemistry coupling of benzoporphyrin derivative (BPD)-Cetuximab photo-immunoconjugates onto FKR560 dye-containing poly(lactic-co-glycolic acid) nanoparticles markedly enhances intracellular photo-immunoconjugate accumulation and potentiates light-activated photo-immunotoxicity in ovarian cancer and glioblastoma. It is further demonstrated that co-delivery and light activation of BPD and FKR560 allow longitudinal fluorescence tracking of photoimmunoconjugate and nanoparticle in cells. Using xenograft mouse models of epithelial ovarian cancer, intravenous injection of photo-immunoconjugated nanoparticles doubles intratumoral accumulation of photo-immunoconjugates, resulting in an enhanced photoimmunotherapy-mediated tumor volume reduction, compared to "standard" immunoconjugates. This generalizable "carrier effect" phenomenon is attributed to the successful incorporation of photo-immunoconjugates onto a nanoplatform, which modulates immunoconjugate delivery and improves treatment outcomes.