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Dynamic interactions within the tumor micro-environment drive patient response to immune checkpoint inhibitors. Existing preclinical models lack true representation of this complexity. Using a Head and Neck cancer patient derived TruTumor histoculture platform, the response spectrum of 70 patients to anti-PD1 treatment is investigated in this study. With a subset of 55 patient samples, multiple assays to characterize T-cell reinvigoration and tumor cytotoxicity are performed. Based on levels of these two response parameters, patients are stratified into five sub-cohorts, with the best responder and non-responder sub-cohorts falling at extreme ends of the spectrum. The responder sub-cohort exhibits high T-cell reinvigoration, high tumor cytotoxicity with T-cells homing into the tumor upon treatment whereas immune suppression and tumor progression pathways are pre-dominant in the non-responders. Some moderate responders benefit from combination of anti-CTLA4 with anti-PD1, which is evident from better cytotoxic T-cell: T-regulatory cell ratio and enhancement of tumor cytotoxicity. Baseline and on-treatment gene expression signatures from this study stratify responders and non-responders in unrelated clinical datasets. Tumor histocultures have been exploited as tools to predict response to cancer therapy. Here the authors report the development and testing of a tumor histoculture platform to study response to immune checkpoint inhibitors in head and neck squamous cell carcinoma.

BackgroundA 3D histo-culture platform provides a near native Tumor immune Micro-Environment (TiME), making it best suited for evaluating response to immunotherapy drugs. Farcast™ TiME is a human 3D tumor histo-culture platform that preserves TiME and maintains functional fidelity of intra-tumoral immune cells (IIC). In this study we investigated the utility of this platform in demonstrating treatment induced Antibody-Dependent Cellular Cytotoxicity (ADCC) mechanism driven by IICs alone versus co-culture with autologous peripheral blood immune cells.MethodsHead and neck squamous cell carcinoma tissue samples (n=5) along with matched blood from consented patients were used in this study. All Peripheral Blood Nucleated Cells (PBNCs) including lymphocytes, monocytes, NK cells and neutrophils were isolated and stained with a tracking dye to distinguish them from IICs. Tumor tissues were processed to generate explants, treated with 184 µg/ml Cetuximab (anti-EGFR) or vehicle control, and cultured with or without PBNCs for 72 hrs. Response was evaluated using flow cytometry and cytokine release assay.ResultsAmount of infiltrated autologous PBNCs showed a strong negative correlation (R2=0.98) with the amount of IICs in the absence of drug treatment. The proportions of infiltrated immune cell sub-populations were similar to the composition of PBNCs added in culture. Cetuximab treatment, however, led to enhanced infiltration of the effector cells for ADCC driven tumor killing, namely NK cells, macrophages, neutrophils, and cytotoxic T cells (CTLs). Notably the unique infiltration pattern of effector cell populations observed in each sample was reflected in the secretion of specific cytokine/chemokines associated with that cell population. NK cell increase (fold change: 1.6 ± 0.8) was observed in all samples with a concomitant increase in MCP-1 secretion (fold change: 1.7 ± 0.9). Granzyme-B expressing NK cells increased (>1.7 fold) in a subset of samples. Samples showing increase in neutrophil infiltration exhibited increased MMP9 secretion, involved in neutrophil infiltration via stromal remodeling. Sample with highest increase in infiltration of CD16+ Monocyte/Macrophages (>2.4 fold) showed maximum increase in Granzyme-B secretion with respect to the untreated arm. Increase in fold secretion (>1.4) of CXCL9/CXCL10 was associated with the sample that showed highest fold increase of Granzyme-B expressing CTL in comparison to untreated arm. IICs alone were not sufficient in eliciting optimal ADCC response.ConclusionsThe study demonstrated ADCC response in the explant/PBNC co-culture platform leading to specific infiltration of effector sub-populations. FarcastTM TiME thus provides a unique platform to explore for heterologous adoptive cell and CAR-T therapies that involve immune cell infiltration.Ethics ApprovalAll samples included in the study were approved by institutional review boards of the centers providing the samples.

In this present study, Mn2+doped CoAl2O4 (MnxCo1−xAl2O4; x = 0.0 to 1.0) spinel nanoparticles were synthesized by microwave combustion method using nitrates of Co, Mn, and Al as the starting materials and urea was used as the fuel. The effects of Mn2+ doping on structural, morphological, opto-magnetic and catalytic properties were studied. Powder X-ray diffraction analysis was confirmed the formation of cubic spinel aluminate structure. Debye-Scherrer’s formula was used to estimate the average crystallite size of the samples and was found to be in the range of 18.26 to 21.47 nm. It was observed that the calculated lattice parameter value is increased from 8.215 to 8.247 Å with increasing the Mn2+ content due to the higher ionic radius of Mn2+ ion. High resolution scanning electron microscopy (HR-SEM) and transmission electron microscopy (HR-TEM) analysis confirmed the nano-sized particle-like morphology of the samples. Energy dispersive X-ray (EDX) results showed the pure form of spinel aluminate structure. The band gap energy (Eg) of undoped CoAl2O4 was estimated to be 3.58 eV from UV-visible diffuse reflectance spectroscopy (DRS), and the Eg values increased with increase of Mn2+ ions due to the decrease of grain size. The magnetic hysteresis (M-H) loop showed the superparamagnetic nature of the samples, and the saturation magnetization values increased with increasing Mn2+ ions, which was confirmed by vibrating sample magnetometer (VSM). All compositions of the samples were successfully tested as catalyst for the conversion of benzyl alcohol into benzaldehyde and observed good catalytic activity.

BackgroundBaseline signatures have so far failed to accurately predict response to immune checkpoint inhibitors. A model that mimics the clinical response of tumor immune microenvironment (TiME) upon treatment in culture, would dramatically improve the chances of identifying true responders. Tumor histocultures capture the complex interactions between various tumor microenvironment components effectively. They retain the immune cell repertoire along with spatial context, both of which are adequately represented in FarcastTM TiME histoculture platform.MethodsFarcastTM head and neck squamous cell carcinoma histoculture platform was used to evaluate response to treatment with anti-PD1 (Nivolumab) alone or in combination with anti-CTLA4 (Ipilimumab) in culture. Multiple assays including spatial analysis was performed post-treatment.ResultsInterferon gamma (INFg) cytokine release, that classically defines T- cell response, was used to stratify samples for response to treatment with Nivolumab (n=45). Histopathological evaluation revealed that 4 of 45 (9%) samples showed an increase in tumor infiltrating immune cells by 1.5 folds, with a concomitant increase in IFNg secretion (Log2FC ± SD,1.67 ± 0.8), and CD8+GranzymeB+ cells estimated by flow cytometry. Three out of these 4 samples showed an increase in tumoricidal response. To capture the complex spatial interplay between TiME components and the intra-cellular signals that determine response, a 72-protein marker GeoMx DSP (nanoString) panel was used on two samples (one recurrent and a primary) treated with Nivolumab. panCK was used for tumor segmentation. An increased expression of FoxP3 (Treg marker), IDO and phosphorylated tumor markers was observed in the recurrent sample within the tumor nest, suggestive of immune suppressive microenvironment post treatment. Interestingly, the primary sample exhibited increase in checkpoint and T-cell costimulatory molecules within the tumor nest along with a significant decrease in FoxP3 and tumor marker EGFR, suggestive of enhanced T- cell mediated tumor cytotoxicity. To map the relative spatial distribution of CD8+ and FoxP3+ cell populations, multiplex IHC assay was performed, and data analyzed using QuPath. Decrease in distance between CD8+ cells from tumor nest with a concomitant increase in distance between FoxP3+ and CD8+ cells were observed in a high IFNg and GranzymeB secreting sample.ConclusionsIn summary, our data suggests that though IFNg secretion was observed in multiple samples post treatment, spatial contexture influenced T-cell response mediated tumor cytotoxicity. Farcast TiME thus provides a unique platform that can combine spatial dynamics of immune cells overlaid with bulk data, providing important insights into understanding underlying mechanisms that counter T-cell response to immune check point inhibitors.AcknowledgementsTechnical support from nanoString for performing the GeoMx DSP experiment and analysis is acknowledged.Ethics ApprovalAll patient samples used in this study was obtained with prior approvals from individual institutional review boards of participating hospitals.