Compared with traditional 2D adherent cell culture, 3D spheroidal cell aggregates, or spheroids, are regarded as more physiological, and this technique has been exploited in the field of oncology, stem cell biology, and tissue engineering. Mesenchymal stem cells (MSCs) cultured in spheroids have enhanced anti-inflammatory, angiogenic, and tissue reparative/regenerative effects with improved cell survival after transplantation. Cytoskeletal reorganization and drastic changes in cell morphology in MSC spheroids indicate a major difference in mechanophysical properties compared with 2D culture. Enhanced multidifferentiation potential, upregulated expression of pluripotency marker genes, and delayed replicative senescence indicate enhanced stemness in MSC spheroids. Furthermore, spheroid formation causes drastic changes in the gene expression profile of MSC in microarray analyses. In spite of these significant changes, underlying molecular mechanisms and signaling pathways triggering and sustaining these changes are largely unknown.

Cell therapy with adult mesenchymal stem cells (MSCs) is a promising approach to regenerative medicine and autoimmune diseases. There are various approaches to improve the efficacy of MSC-based therapeutics, and MSC preparation as spheroidal aggregates, or MSC spheroids, is a novel preparatory and delivery method. Spheroid formation induces a dramatic change in the gene expression profile of MSCs. Self-activation of interleukin-1 (IL1) signaling was shown to be upstream of both pro-and anti-inflammatory genes in MSC spheroids, but the molecular pathways that initiate IL1 signaling remain unknown. As bone morphogenic protein (BMP)2 upregulation precedes that of IL1B expression during spheroid formation, we hypothesized that BMP2 signaling triggers IL1 signaling in MSC spheroids. Contrary to expectations, BMP2 signaling decreased expression of IL1B and downstream genes in a SMAD6-dependent manner. Conversely, IL1B signaling enhanced BMP2 expression. Another major difference between two-dimensional (2D) monolayer culture and three-dimensional (3D) spheroid culture is the Young's elasticity modulus, or stiffness, of the materials surrounding the cells, as there is a million-fold difference between a plastic surface for standard 2D culture (GPa) and 3D spheroidal aggregates (0.1 kPa). We tested another hypothesis that soft elasticity-associated mechanosignaling initiates the gene expression change during spheroid formation. Results showed that both BMP2 expression and inflammatory signaling are upregulated in an elasticity-associated signaling-dependent manner in MSCs. Lastly, BMP2 signaling enhanced cell survival and cell spreading of MSC spheroids. In summary, our study suggests that soft elasticity and BMP2 signaling are critical for MSC spheroids.

CRISPR/Cas systems coupled with reverse transcriptase (RT), such as the recently described Prime editing, allow for site-specific replacement of DNA sequences. Despite widespread testing of Prime editing, it is currently only compatible with type II CRISPR/Cas proteins such as Streptococcus pyogenes and Staphylococcus aureus Cas9. Enabling RT compatibility with other CRISPR/Cas domains, such as type V enzymes with orthogonal protospacer adjacent motif specificities and smaller protein size would expand the range of edits that can be made in therapeutic and industrial applications. We achieve this with a novel mode of DNA editing at CRISPR-targeted sites that reverse transcribes the edit into the target strand DNA (e.g., the complement of the PAM-containing strand), rather than the non-target strand DNA, as in Prime editing. We term this technology RNA encoded DNA replacement of alleles with CRISPR (hereafter, REDRAW). We show that REDRAW extends the utility of RT-mediated editing beyond type II to include multiple type V CRISPR domains. REDRAW features a broad (8-10 bases) targeting window, at which all types of substitutions, insertions and deletions are possible. REDRAW combines the advantages of type V CRISPR domains with the extensive range of genetic variation enabled by RT-mediated, templated sequence replacement strategies.

Introduction: It has been proposed that the presence of inflamed tumor phenotypes, characterized by the presence of infiltrating lymphocytes and the expression of specific chemokines and cytokines, can predict response to immunotherapy and result in better patient outcomes [1, 2]. We hypothesized that pelareorep, an immuno-oncolytic virus (IOV), may elicit predictive proinflammatory gene signatures in select cancer cell lines permissive to viral infection. Methods: Cell lines derived from non-small cell lung cancer (NSCLC, H522), colorectal cancer (CRC, SW-620), and hepatocellular carcinoma (HCC, SNU 387) were infected at an multiplicity of infection equal to 50. We examined changes in gene expression and conducted cell viability assays at 6, 12, and 18 hours post pelareorep infection (including a non-infected control). To monitor changes in gene expression we employed a custom 780-gene Pan Cancer Immune panel developed by nanoString Technologies and specifically monitored for changes in the expression of key interferon and NF-κB signalling genes, immune checkpoint ligands, and a 12-gene chemokine signature predictive of a positive response to immunotherapy identified by Messina et al. [2]. Results: All cell lines examined were susceptible to pelareorep induced cytopathic effect. Strikingly, principal component analysis revealed that the changes in gene expression were unique and different for each cell line. Of the cell lines examined, only HCC cells infected with pelareorep promoted an inflammatory signature, similar to the one used to predict response to immunotherapy in melanoma [2]. Conclusions: This study demonstrates that pelareorep can prime or promote a predictive inflamed tumor phenotype in HCC, which correlates with the innate response recently described in HCC- animal models treated with pelareorep [3]. The role of pelareorep in the treatment of hepatocellular carcinoma deserves further investigation, particularly in combination with other immunotherapies. References: [1] Gajewski, T.F., The Next Hurdle in Cancer Immunotherapy: Overcoming the Non-T-Cell-Inflamed Tumor Microenvironment. Semin Oncol, 2015. 42(4): p. 663-71. [2] Messina, J.L., et al., 12-Chemokine gene signature identifies lymph node-like structures in melanoma: potential for patient selection for immunotherapy? Sci Rep, 2012. 2: p. 765. [3] Samson, A., et al., Oncolytic reovirus as a combined antiviral and anti-tumour agent for the treatment of liver cancer. Gut, 2016. Citation Format: Grey A. Wilkinson, Aine Piar, Zoe Cesarz, Hue Tran, Romit Chakrabarty, Andres Gutierrez, Matt Coffey. Pelareorep promotes the expression of a chemokine signature that predicts response to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4707.