LncRNA FENDRR Expression Correlates with Tumor Immunogenicity.

FENDRR (Fetal-lethal non-coding developmental regulatory RNA, LncRNA FOXF1-AS1) is a recently identified tumor suppressor long non-coding (LncRNA) RNA, and its expression has been linked with epigenetic modulation of the target genes involved in tumor immunity. In this study, we aimed to understand the role of FENDRR in predicting immune-responsiveness and the inflammatory tumor environment. Briefly, FENDRR expression and its relationship to immune activation signals were assessed in murine cell lines. Data suggested that tumor cells (e.g., C26 colon, 4T1 breast) that typically upregulate immune activation genes and the MHC class I molecule exhibited high FENDRR expression levels. Conversely, tumor cells with a generalized downregulation of immune-related gene expression (e.g., B16F10 melanoma) demonstrated low to undetectable FENDRR levels. Mechanistically, the modulation of FENDRR expression enhanced the inflammatory and WNT signaling pathways in tumors. Our early data suggest that FENDRR can play an important role in the development of immune-relevant phenotypes in tumors, and thereby improve cancer immunotherapy.

Boiling histotripsy and in-situ CD40 stimulation improve the checkpoint blockade therapy of poorly immunogenic tumors.

Background: Advanced stage cancers with a suppressive tumor microenvironment (TME) are often refractory to immune checkpoint inhibitor (ICI) therapy. Recent studies have shown that focused ultrasound (FUS) TME-modulation can synergize ICI therapy, but enhancing survival outcomes in poorly immunogenic tumors remains challenging. Here, we investigated the role of focused ultrasound based boiling histotripsy (HT) and in-situ anti-CD40 agonist antibody (αCD40) combinatorial therapy in enhancing therapeutic efficacy against ICI refractory murine melanoma. Methods: Unilateral and bilateral large (~330-400 mm3) poorly immunogenic B16F10 melanoma tumors were established in the flank regions of mice. Tumors were exposed to single local HT followed by an in-situ administration of αCD40 (HT+ αCD40: HT40). Inflammatory signatures post treatment were assessed using pan-cancer immune profiling and flow cytometry. The ability of HT40 ± ICI to enhance local and systemic effects was determined by immunological characterization of the harvested tissues, and by tumor growth delay of local and distant untreated tumors 4-6 weeks post treatment. Results: Immune profiling revealed that HT40 upregulated a variety of inflammatory markers in the tumors. Immunologically, HT40 treated tumors showed an increased population of granzyme B+ expressing functional CD8+ T cells (~4-fold) as well as an increased M1 to M2 macrophage ratio (~2-3-fold) and CD8+ T: regulatory T cell ratio (~5-fold) compared to the untreated control. Systemically, the proliferation rates of the melanoma-specific memory T cell population were significantly enhanced by HT40 treatment. Finally, the combination of HT40 and ICI therapy (anti-CTLA-4 and anti-PD-L1) caused superior inhibition of distant untreated tumors, and prolonged survival rates compared to the control. Conclusions: Data suggest that HT40 reprograms immunologically cold tumors and sensitizes them to ICI therapy. This approach may be clinically useful for treating advanced stage melanoma cancers.

Reprogramming the rapid clearance of thrombolytics by nanoparticle encapsulation and anchoring to circulating red blood cells.

Rapid clearance of thrombolytics from blood following intravenous injection is a major clinical challenge in cardiovascular medicine. To overcome this barrier, nanoparticle (NP) based drug delivery systems have been reported. Although superior than conventional therapy, a large proportion of the injected NP is still cleared by the reticuloendothelial system. Previously, we and others showed that ex vivo attachment of bioscavengers, thrombolytics, and nanoparticles (NPs) to glycophorin A receptors on red blood cells (RBCs) improved the blood half-life. This is promising, but ex-vivo approaches are cumbersome and challenging to translate clinically. Here, we developed a novel Ter119-polymeric NP containing tissue plasminogen activator for on-demand targeting of GPA receptors in vivo. Upon intravenous injection, the Ter119-NPs achieved remarkable RBC labeling efficiencies (>95%), resulting in marked enhancement of blood residence time of tPA from minutes to several days without any morphological, hematological, and histological complications. Our approach of RBC labeling with the NPs also prevented reticuloendothelial detections and the activations of innate and adaptive immune system. Data suggest that real-time targeting of therapeutics to RBC with NPs can potentially improve outcomes and reduce complications against a variety chronic disease.

Novel calreticulin-nanoparticle in combination with focused ultrasound induces immunogenic cell death in melanoma to enhance antitumor immunity.

Rationale: Some studies have shown that the local activation of immunogenic cell death (ICD) by upregulating calreticulin (CRT) expression in solid tumors can improve antitumor effects. Although a promising approach, a key current challenge in ICD tumor therapy is the absence of a clinically translatable method for reproducibly inducing the CRT expression. Herein, we report a novel calreticulin-nanoparticle (CRT-NP) that enhances ICD and synergizes with focused ultrasound (FUS) to achieve local and systemic antitumor effects. Methods: Full-length clone DNA of calreticulin was encapsulated in NPs made from DOTAP and cholesterol. Three CRT-NP intratumoral injections of 20 µg each were given 2 days apart, and FUS heating (42-45°C, ~15min) was applied sequentially 24h after each injection to induce ICD. To investigate ICD specific immune effect, the splenocytes of mice vaccinated with CRT-NP (± FUS) treated B16F10 cells were evaluated ex-vivo for TRP-2 antigen specific immunity. Additionally, the long-term protection was evaluated by re-challenging with the melanoma cells in the flank regions of tumor bearing mice. Results: CRT-NP plus FUS (CFUS) upregulated CRT expression, expanded the population of melanoma TRP-2 specific functional CD4+ and CD8+ T cells and tumor-suppressing M1 phenotype, and increased PD-1 and PD-L1 marker expression in the T cells. Therapeutically, CFUS suppressed B16 melanoma growth by >85% vs. that seen in untreated controls, and >~50% vs. CRT-NP or FUS alone, and prevented tumor growth in distal untreated sites. Conclusions: CRT-NP amplifies the FUS and ICD therapeutic outcomes against melanoma, suggesting that the proposed combinatorial methodology may be clinically translatable.

In-situ vaccination using focused ultrasound heating and anti-CD-40 agonistic antibody enhances T-cell mediated local and abscopal effects in murine melanoma.

The success of melanoma immunotherapy is dependent on the presence of activated and functional T-cells in tumors. The objective of this study was to investigate the impact of local-focused ultrasound (FUS) heating (∼42-45 °C) and in-situ anti-CD-40 agonistic antibody in enhancing T-cell function for melanoma immunotherapy. We compared the following groups of mice with bilateral flank B16 F10 melanoma: (1) Control, (2) FUS, (3) CD-40, and (4) CD-40 + FUS (FUS40). FUS heating was applied for ∼15 min in right flank tumor, and intratumoral injections of CD-40 were performed sequentially within 4 h. A total of 3 FUS and 4 anti-CD-40 treatments were administered unilaterally 3 days apart. Mice were sacrificed 30 days post-inoculation, and the treated tumor and spleen tissues were profiled for T-cell function and macrophage polarization. Compared to all other groups, histology and flow cytometry showed that FUS40 increased the population of tumor-specific CD-4+ and CD-8+ T cells rich in Granzyme B+, interleukin-2 (IL-2) and IFN-γ production and poor in PD-1 expression. In addition, FUS40 promoted the infiltration of tumor-suppressing M1 phenotype macrophages in the treated mice. The resultant immune-enhancing effects of FUS40 suppressed B16 melanoma growth at the treated site by 2-3-folds compared to control, FUS, and CD-40, and also achieved significant abscopal effects in untreated tumors relative to CD40 alone. Additionally, the local FUS40 prevented adverse liver toxicities in the treated mice. Our study suggests that combined FUS and CD-40 can enhance T-cell and macrophage functions to aid effective melanoma immunotherapy.