Currently approved inhibitors of the PD-1/PD-L1 pathway represent a major advance for the treatment of lung cancers, yet they are ineffective in a majority of patients due to lack of preexisting T-cell reactivity. Here, we show that a TLR9 agonist delivered by inhalation is able to prime T-cell responses against poorly immunogenic lung tumors and to complement the effects of PD-1 blockade. Inhaled TLR9 agonist causes profound remodeling in tumor-bearing lungs, leading to the formation of tertiary lymphoid structures adjacent to the tumors, CD8 T-cell infiltration into the tumors, dendritic cell expansion, and antibody production. Inhalation of TLR9 agonist also increased the pool of functional PD-1T-bet effector CD8 T cells in tumor-bearing lungs. Effector CD8 T cells generated by inhaled TLR9 agonist treatment were licensed by PD-1 blockade to become highly functional CTLs, leading to a durable rejection of both lung tumors and tumor lesions outside the lungs. CD4 T cells activated in response to inhaled TLR9 play a critical role in this process by controlling the proliferation, preventing exhaustion, and guiding the differentiation of optimally functional CTLs. This study characterizes a strategy to apply localized TLR9 stimulation to a tumor type not accessible for direct injection, a strategy that may expand the therapeutic potential of PD-1 blockade in non-small cell lung cancer. These findings demonstrate that local delivery of a toll-like receptor 9 agonist can change the immune content of an entire organ and enhance the efficacy of immune checkpoint inhibition. http://cancerres.aacrjournals.org/content/canres/78/17/4943/F1.large.jpg .

Microproteomic studies have improved our knowledge of cell biology. Yet, with mass spectrometry (MS) analysis, accuracy can be lost for protein identification and quantification when using heterogeneous samples. Laser capture microdissection (LCM) allows for the enrichment of specific subsets of cells to study their proteome; however, sample fixation is necessary. Unfortunately, fixation hampers MS results due to protein cross-linking. The aim of this study was to identify both a fixation protocol and an extraction method that returns the best yield of proteins for downstream MS analysis, while preserving cellular structures. We compared glutaraldehyde (GLU), a common fixative to preserve cells, to dithiobispropionimidate (DTBP), a cleavable cross-linker. Our DTBP fixation/extraction protocol greatly increased the protein recovery. In fact, while 1000 GLU fixed cells returned only 159 unique protein hits, from 1464 unique peptides of 1994 unique collected spectra, 1000 DTBP fixed cells resulted in 567 unique collected protein hits, from 7542 unique peptides, of 10,401 unique collected spectra. That is, a 3.57-fold increase in protein hits, 5.15-fold increase in unique peptides, and a 5.22-fold increase in unique collected spectra. Overall, the novel protocol introduced here allows for a very efficient protein recovery and good sample quality for MS after sample collection using LCM.

Cell–cell communication is vital to multicellular organisms, and distinct types of cellular protrusions play critical roles during development, cell signaling, and the spreading of pathogens and cancer. The differences in the structure and protein composition of these different types of protrusions and their specific functions have not been elucidated due to the lack of a method for their specific isolation and analysis. In this paper, we described, for the first time, a method to specifically isolate distinct protrusion subtypes, based on their morphological structures or fluorescent markers, using laser capture microdissection (LCM). Combined with a unique fixation and protein extraction protocol, we pushed the limits of microproteomics and demonstrate that proteins from LCM-isolated protrusions can successfully and reproducibly be identified by mass spectrometry using ultra-high field Orbitrap technologies. Our method confirmed that different types of protrusions have distinct proteomes and it promises to advance the characterization and the understanding of these unique structures to shed light on their possible role in health and disease.

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In cancer settings severe T cell exhaustion is one major road block limiting immunotherapy efficacy. Epigenetic changes happening during T cell effector differentiation lock T cells in a non-programmable state which limits the efficacy of checkpoint blockade. In addition, histone modification has been reported to critically modulate MHC I and II expression on tumor cells and influx of T cells into tumor. We have previously shown that intratumoral administration of a TLR9 agonist, SD-101, induces T cell responses capable of attacking tumor lesions throughout the body. SD-101, combined with the PD-1 inhibitor pembrolizumab, is currently being investigated in patients with metastatic melanoma and head and neck cancer with encoraging results. Domatinostat (4SC AG) is a well-tolerated orally available selective class I histone deacetylase inhibitor HDACi) with demonstrated anti-tumor activity in hematological cancers as well as in numerous animal models of solid tumors as monotherapy and in combination with checkpoint blockade. Domatinostat is currently being investigated in patients with metastatic melanoma and gastrointestinal cancers in combination with PD(L)-1 antibodies. This study addresses whether the addition of domatinostat to the SD-101 regimen would potentiate the T cell response induced by SD-101. Studies were conducted in multiple syngeneic models in which tumors grow in multiple site of the body but only one is treated intratumorally with SD-101 and domatinostat is given systemically (oral). SD-101 and domatinostat were given alone or in combination, at clinically relevant dosages. The combination of intratumoral SD-101 and domatinostat induced substantial regression of the primary tumor (injected) and distant site cutaneous tumors, as well as lung metastases The combination was superior to either agent given alone and led to increased tumor specific cytotoxic CD8 T cells (CTL) infiltrating distant site lesions. CTL’s were characterized by a low exhaustion phenotype, increased proliferative capacity and increased co-expression of cytolytic markers, CD107 and GranzymeB. Gene expression profiling was performed on abscopal tumors using the PanCancer immune profiling panel from Nanostring. The combination of SD-101 and domatinostat led to upregulation of multiple immune related signatures indicating increased T, NK and dendritic cell functions. Notably, the combination of SD-101 and domatinostat was more efficacious than SD-101 combined with various other epigenetic compounds. The addition of PD-1 blockade to SD-101 and domatinostat further boosted T cell responses leading to rejection in mice with high metastatic burdens. These results suggest that domatinostat acts on SD-101 primed tumor specific CD8+ T cells to expand them, render them more cytolytic and facilitate their infiltration in abscopal tumors. Citation Format: Émilie Degagné, Jose Romo, Marilena Gallotta, Shravan Kannan, Robert L. Coffman, Cristiana Guiducci. Tumor abscopal responses induced by the TLR9 agonist, SD-101, are strongly potentiated by a HDAC class I inhibitor, domatinostat [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2259.