Dual-Function Antibody Conjugate-Enabled Photoimmunotherapy Complements Fluorescence and Photoacoustic Imaging of Head and Neck Cancer Spheroids.

Several molecular-targeted imaging and therapeutic agents are in clinical trials for image-guided surgery and photoimmunotherapy (PIT) for head and neck cancers. In this context, we have previously reported the development, characterization, and specificity of a dual-function antibody conjugate (DFAC) for multimodal imaging and photoimmunotherapy (PIT) of EGFR-overexpressing cancer cells. The DFAC reported previously and used in the present study comprises an EGFR-targeted antibody, cetuximab, conjugated to benzoporphyrin derivative (BPD) for fluorescence imaging and PIT and a Si-centered naphthalocyanine dye for photoacoustic imaging. We report here the evaluation and performance of DFAC in detecting microscopic cancer spheroids by fluorescence and photoacoustic imaging along with their treatment by PIT. We demonstrate that while fluorescence imaging can detect spheroids with volumes greater than 0.049 mm3, photoacoustic imaging-based detection was possible even for the smallest spheroids (0.01 mm3) developed in the study. When subjected to PIT, the spheroids showed a dose-dependent response, with smaller spheroids (0.01 and 0.018 mm3) showing a complete response with no recurrence when treated with 100 J/cm2. Together our results demonstrate the complementary imaging and treatment capacity of DFAC. This potentially enables fluorescence imaging to assess the presence of tumor on a macroscopic scale, followed by photoacoustic imaging for delineating tumor margins guiding surgical resection and elimination of any residual microscopic disease by PIT, in a single intraoperative setting.

A Dual Function Antibody Conjugate Enabled Photoimmunotherapy Complements Fluorescence and Photoacoustic Imaging of Head and Neck Cancer Spheroids.

Several molecular-targeted imaging and therapeutic agents are in clinical trials for image-guided surgery and photoimmunotherapy (PIT) of head and neck cancers. In this context, we have previously reported the development, characterization, and specificity of a dual function antibody conjugate (DFAC) for multi-modal imaging and photoimmunotherapy (PIT) of EGFR over-expressing cancer cells. The DFAC reported previously and used in the present study, comprises of an EGFR targeted antibody - Cetuximab conjugated to Benzoporphyrin derivative (BPD) for fluorescence imaging and PIT, and a Si-centered naphthalocyanine dye for photoacoustic imaging. We report here the evaluation and performance of DFAC in detecting microscopic cancer spheroids by fluorescence and photoacoustic imaging along with their treatment by PIT. We demonstrate that while fluorescence imaging can detect spheroids with volumes greater than 0.049 mm3, photoacoustic imaging-based detection was possible even for the smallest spheroids (0.01 mm3), developed in the study. When subjected to PIT, the spheroids showed a dose-dependent response with smaller spheroids (0.01 and 0.018 mm3) showing a complete response with no recurrence when treated with 100 J/cm2. Together our results demonstrate the complementary imaging and treatment capacity of DFAC. This potentially enables fluorescence imaging to assess tumor presence on a macroscopic scale followed by photoacoustic imaging for delineating tumor margins guiding surgical resection and elimination of any residual microscopic disease by PIT, in a single intra-operative setting.

Photoimmunotherapy Retains Its Anti-Tumor Efficacy with Increasing Stromal Content in Heterotypic Pancreatic Cancer Spheroids.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by increased levels of desmoplasia that contribute to reduced drug delivery and poor treatment outcomes. In PDAC, the stromal content can account for up to 90% of the total tumor volume. The complex interplay between stromal components, including pancreatic cancer-associated fibroblasts (PCAFs), and PDAC cells in the tumor microenvironment has a significant impact on the prognoses and thus needs to be recapitulated in vitro when evaluating various treatment strategies. This study is a systematic evaluation of photodynamic therapy (PDT) in 3D heterotypic coculture models of PDAC with varying ratios of patient-derived PCAFs that simulate heterogeneous PDAC tumors with increasing stromal content. The efficacy of antibody-targeted PDT (photoimmunotherapy; PIT) using cetuximab (a clinically approved anti-EGFR antibody) photoimmunoconjugates (PICs) of a benzoporphyrin derivative (BPD) is contrasted with that of liposomal BPD (Visudyne), which is currently in clinical trials for PDT of PDAC. We demonstrate that both Visudyne-PDT and PIT were effective in heterotypic PDAC 3D spheroids with a low stromal content. However, as the stromal content increases above 50% in the 3D spheroids, the efficacy of Visudyne-PDT is reduced by up to 10-fold, while PIT retains its efficacy. PIT was found to be 10-, 19-, and 14-fold more phototoxic in spheroids with 50, 75, and 90% PCAFs, respectively, as compared to Visudyne-PDT. This marked difference in efficacy is attributed to the ability of PICs to penetrate and distribute homogeneously within spheroids with a higher stromal content and the mechanistically different modes of action of the two formulations. This study thus demonstrates how the stromal content in PDAC spheroids directly impacts their responsiveness to PDT and proposes PIT to be a highly suited treatment option for desmoplastic tumors with particularly high degrees of stromal content.

The essential polysome-associated RNA-binding protein RBP42 targets mRNAs involved in Trypanosoma brucei energy metabolism.

RNA-binding proteins that target mRNA coding regions are emerging as regulators of post-transcriptional processes in eukaryotes. Here we describe a newly identified RNA-binding protein, RBP42, which targets the coding region of mRNAs in the insect form of the African trypanosome, Trypanosoma brucei. RBP42 is an essential protein and associates with polysome-bound mRNAs in the cytoplasm. A global survey of RBP42-bound mRNAs was performed by applying HITS-CLIP technology, which captures protein-RNA interactions in vivo using UV light. Specific RBP42-mRNA interactions, as well as mRNA interactions with a known RNA-binding protein, were purified using specific antibodies. Target RNA sequences were identified and quantified using high-throughput RNA sequencing. Analysis revealed that RBP42 bound mainly within the coding region of mRNAs that encode proteins involved in cellular energy metabolism. Although the mechanism of RBP42's function is unclear at present, we speculate that RBP42 plays a critical role in modulating T. brucei energy metabolism.

PolyA-specific ribonuclease (PARN-1) function in stage-specific mRNA turnover in Trypanosoma brucei.

Deadenylation is often the rate-limiting event in regulating the turnover of cellular mRNAs in eukaryotes. Removal of the poly(A) tail initiates mRNA degradation by one of several decay pathways, including deadenylation-dependent decapping, followed by 5' to 3' exonuclease decay or 3' to 5' exosome-mediated decay. In trypanosomatids, mRNA degradation is important in controlling the expression of differentially expressed genes. Genomic annotation studies have revealed several potential deadenylases. Poly(A)-specific RNase (PARN) is a key deadenylase involved in regulating gene expression in mammals, Xenopus oocytes, and higher plants. Trypanosomatids possess three different PARN genes, PARN-1, -2, and -3, each of which is expressed at the mRNA level in two life-cycle stages of the human parasite Trypanosoma brucei. Here we show that T. brucei PARN-1 is an active deadenylase. To determine the role of PARN-1 on mRNA stability in vivo, we overexpressed this protein and analyzed perturbations in mRNA steady-state levels as well as mRNA half-life. Interestingly, a subset of mRNAs was affected, including a family of mRNAs that encode stage-specific coat proteins. These data suggest that PARN-1 functions in stage-specific protein production.