Single cell metabolic imaging of tumor and immune cells in vivo in melanoma bearing mice

IntroductionMetabolic reprogramming of cancer and immune cells occurs during tumorigenesis and has a significant impact on cancer progression. Unfortunately, current techniques to measure tumor and immune cell metabolism require sample destruction and/or cell isolations that remove the spatial context. Two-photon fluorescence lifetime imaging microscopy (FLIM) of the autofluorescent metabolic coenzymes nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) provides in vivo images of cell metabolism at a single cell level.MethodsHere, we report an immunocompetent mCherry reporter mouse model for immune cells that express CD4 either during differentiation or CD4 and/or CD8 in their mature state and perform in vivo imaging of immune and cancer cells within a syngeneic B78 melanoma model. We also report an algorithm for single cell segmentation of mCherry-expressing immune cells within in vivo images.ResultsWe found that immune cells within B78 tumors exhibited decreased FAD mean lifetime and an increased proportion of bound FAD compared to immune cells within spleens. Tumor infiltrating immune cell size also increased compared to immune cells from spleens. These changes are consistent with a shift towards increased activation and proliferation in tumor infiltrating immune cells compared to immune cells from spleens. Tumor infiltrating immune cells exhibited increased FAD mean lifetime and increased protein-bound FAD lifetime compared to B78 tumor cells within the same tumor. Single cell metabolic heterogeneity was observed in both immune and tumor cells in vivo.DiscussionThis approach can be used to monitor single cell metabolic heterogeneity in tumor cells and immune cells to study promising treatments for cancer in the native in vivo context

Antibody landscape of C57BL/6 mice cured of B78 melanoma via a combined radiation and immunocytokine immunotherapy regimen

Sera of immune mice that were previously cured of their melanoma through a combined radiation and immunocytokine immunotherapy regimen consisting of 12 Gy of external beam radiation and the intratumoral administration of an immunocytokine (anti-GD2 mAb coupled to IL-2) with long-term immunological memory showed strong antibody-binding against melanoma tumor cell lines via flow cytometric analysis. Using a high-density whole-proteome peptide array (of 6.090.593 unique peptides), we assessed potential protein-targets for antibodies found in immune sera. Sera from 6 of these cured mice were analyzed with this high-density, whole-proteome peptide array to determine specific antibody-binding sites and their linear peptide sequence. We identified thousands of peptides that were targeted by these 6 mice and exhibited strong antibody binding only by immune (after successful cure and rechallenge), not naïve (before tumor implantation) sera and developed a robust method to detect these differentially targeted peptides. Confirmatory studies were done to validate these results using 2 separate systems, a peptide ELISA and a smaller scale peptide array utilizing a slightly different technology. To the best of our knowledge, this is the first study of the full set of germline encoded linear peptide-based proteome epitopes that are recognized by immune sera from mice cured of cancer via radio-immunotherapy. We furthermore found that although the generation of B-cell repertoire in immune development is vastly variable, and numerous epitopes are identified uniquely by immune serum from each of these 6 immune mice evaluated, there are still several epitopes and proteins that are commonly recognized by at least half of the mice studied. This suggests that every mouse has a unique set of antibodies produced in response to the curative therapy, creating an individual “fingerprint.” Additionally, certain epitopes and proteins stand out as more immunogenic, as they are recognized by multiple mice in the immune group

Results of a collaborative study on DNA identification of aged bone samples

AimA collaborative exercise with several institutes was organized by the Forensic DNA Service (FDNAS) and the Institute of the Legal Medicine, 2nd Faculty of Medicine, Charles University in Prague, Czech Republic, with the aim to test performance of different laboratories carrying out DNA analysis of relatively old bone samples. MethodsEighteen laboratories participating in the collaborative exercise were asked to perform DNA typing of two samples of bone powder. Two bone samples provided by the National Museum and the Institute of Archaelogy in Prague, Czech Republic, came from archeological excavations and were estimated to be approximately 150 and 400 years old. The methods of genetic characterization including autosomal, gonosomal, and mitochondrial markers was selected solely at the discretion of the participating laboratory. ResultsAlthough the participating laboratories used different extraction and amplification strategies, concordant results were obtained from the relatively intact 150 years old bone sample. Typing was more problematic with the analysis of the 400 years old bone sample due to poorer quality. ConclusionThe laboratories performing identification DNA analysis of bone and teeth samples should regularly test their ability to correctly perform DNA-based identification on bone samples containing degraded DNA and potential inhibitors and demonstrate that risk of contamination is minimized

Intratumoral injection reduces toxicity and antibody-mediated neutralization of immunocytokine in a mouse melanoma model

Background Some patients with cancer treated with anticancer monoclonal antibodies (mAbs) develop antidrug antibodies (ADAs) that recognize and bind the therapeutic antibody. This response may neutralize the therapeutic mAb, interfere with mAb effector function or cause toxicities. We investigated the potential influence of ADA to modify the tumor-binding capability of a tumor-reactive ‘immunocytokine’ (IC), namely, a fusion protein (hu14.18-IL2) consisting of a humanized, tumor-reactive, anti-GD2 mAb genetically linked to interleukin 2. We characterize the role of treatment delivery of IC (intravenous vs intratumoral) on the impact of ADA on therapeutic outcome following IC treatments in an established antimelanoma (MEL) regimen involving radiotherapy (RT) +IC.Methods C57BL/6 mice were injected with human IgG or the hu14.18-IL2 IC to develop a mouse anti-human antibody (MAHA) response (MAHA+). In vitro assays were performed to assess ADA binding to IC using sera from MAHA+ and MAHA− mice. In vivo experiments assessed the levels of IC bound to tumor in MAHA+ and MAHA− mice, and the influence of IC route of delivery on its ability to bind to B78 (GD2+) MEL tumors.Results MAHA is inducible in C57BL/6 mice. In vitro assays show that MAHA is capable of inhibiting the binding of IC to GD2 antigen on B78 cells, resulting in impaired ADCC mediated by IC. When B78-bearing mice are injected intravenously with IC, less IC binds to B78-MEL tumors in MAHA+ mice than in MAHA− mice. In contrast, when IC is injected intratumorally in tumor-bearing mice, the presence of MAHA does not detectibly impact IC binding to the tumor. Combination therapy with RT+IT-IC showed improved tumor regression compared with RT alone in MAHA+ mice. If given intratumorally, IC could be safely readministered in tumor-bearing MAHA+ mice, while intravenous injections of IC in MAHA+ mice caused severe toxicity. Histamine levels were elevated in MAHA+ mice compared with MAHA− mice after reintroduction of IC.Conclusions Intratumoral injection may be a means of overcoming ADA neutralization of therapeutic activity of tumor-reactive mAbs or ICs and may reduce systemic toxicity, which could have significant translational relevance

43 Intravital multiphoton imaging of infiltrating CD8 T cell and tumor cell metabolism during immunotherapy in a murine melanoma model

BackgroundIntravital multiphoton microscopy (IMM) provides single cell imaging within intact living systems. IMM of the autofluorescent metabolic co-enzymes NAD(P)H and FAD, optical metabolic imaging (OMI), provides in vivo label-free imaging of metabolic changes. The metabolism of tumor and immune cells is closely associated with cancer progression and tissue site,1–4 so we aim to study metabolic trends during administration of an effective, triple-combination immunotherapy within murine melanoma tumors.5 This therapy includes external beam radiation, intratumoral hu14.18-IL2 immunocytokine (anti-GD2 mAb fused to IL2), and intraperitoneal anti-CTLA-4 leading to in situ vaccination and cure of GD2+ murine tumors.5 Previous work has shown that a T cell response is critical to the efficacy of this therapy,5–6 so we created an mCherry-labeled T cell mouse model to study this response. Here, IMM was used to image concurrent tumor and CD8+ T cell metabolic trends during administration of immunotherapy.MethodsWe created an mCherry-labeled CD8+ T cell mouse model through CRISPR/Cas9 knock-in. We then implanted syngeneic B78 (GD2+) melanoma cells into the flanks of these reporter mice to induce measurable tumors. Mice were anesthetized, skin flap surgery performed, and tumors imaged at several time points. IMM was performed using 750–1040 nm to excite NAD(P)H, FAD, and mCherry through a 40X (1.15 NA) objective. Fluorescence lifetime data was collected using time correlated single photon counting electronics. Murine tissues were harvested and analyzed via flow cytometry and multiplex immunofluorescence to corroborate IMM findings and characterize the immune infiltrate.ResultsHere we demonstrate the feasibility of our IMM platform to capture single cell metabolic changes during immunotherapy administration. Through our intravital imaging we show that CD8 T cell and tumor cell redox ratio (intensity of NAD(P)H/intensity of NAD(P)H + FAD) is significantly increased in treated compared to control mice (figure 1), possibly indicating increased glycolytic activity. We also show differences in protein binding within both CD8 T cells and tumor cells during treatment. Overall, this technology enables analysis of metabolic changes in CD8 T cells and tumor cells in vivo during administration of our immunotherapy regimen.Abstract 43 Figure 1In vivo multiphoton images of immune and tumor cell populations during immunotherapy. A) Representative in vivo fluorescence intensity images of B78 melanoma tumors growing in control and treated CD8 mCherry reporter mice show mCherry-labeled CD8+ T cells (red) infiltrating tumor tissue as well as autofluorescent metabolic coenzymes NAD(P)H (blue) and FAD (green) expressed by the tumor and T cells. Scale bar 25 μm. B) Corresponding in vivo optical redox ratio intensity images show redox balance within the tumor microenvironment. Treated tumors exhibit an increased optical redox ratio which may indicate increased glycolytic activity during immunotherapy. C) Quantified single-cell B78 tumor (n = 353) and CD8 T cell (n = 18) autofluorescence data. Both CD8 T cells (p&lt;0.0001) and tumor cells (p&lt;0.0034) exhibit significantly increased optical redox ratio with treatment (n = 2 mice, median – center bold dashed line, 3rd quartile – upper dashed line, 1st quartile – lower dashed line, Mann-Whitney U Test).ConclusionsThese results provide additional support that the combination of intravital imaging with OMI allows for concurrent imaging of T cell infiltration and metabolic trends. Specifically, OMI enabled us to probe single cell metabolic changes occurring during our immunotherapy regimen. With continued work, this imaging platform may be leveraged to develop new combinations of immunotherapies.AcknowledgementsThis work is supported by the Morgridge Institute for Research (Interdisciplinary Fellowship awarded to A.R.H.) and the NIH (R01 CA205101 and R35 CA197078). The authors thank the University of Wisconsin Carbone Cancer Center (UWCCC) Support Grant P30 CA014520, the UWCCC Translational Research Initiatives in Pathology laboratory - supported by the UW Department of Pathology and Laboratory Medicine and the Office of The Director NIH (S10OD023526), the UWCCC Flow Cytometry Laboratory, and the Genome Editing and Animal Models Laboratory for core services. The authors also thank Tiffany M. Heaster for training and thoughtful discussions as well as Dan Pham for cell isolation help.ReferencesRenner K, Singer K, Koehl GE, Geissler EK, Peter K, Siska P J, Kreutz M. Metabolic Hallmarks of Tumor and Immune Cells in the Tumor Microenvironment. Front Immunol 2017, 8 Mar: 1–11.Mockler MB, Conroy MJ, Lysaght J. Targeting T Cell Immunometabolism for Cancer Immunotherapy; Understanding the Impact of the Tumor Microenvironment. Front Oncol 2014, 4 May: 1–11.Ghesquière B, Wong BW, Kuchnio A, Carmeliet P. Metabolism of stromal and immune cells in health and disease. Nature 2014; 511(7508):167–176.Heaster TM, Heaton AR, Sondel PM, Skala MC. Intravital metabolic autofluorescence imaging captures macrophage heterogeneity across normal and cancerous tissue. Front Bioeng Biotechnol 2021, 9(April): 1–10.Morris ZS, Guy EI, Francis DM, Gressett MM, Werner LR, Carmichael LL, Yang RK, Armstrong EA, Huang S, Navid F, Gillies SD, Korman A, Hank JA, Rakhmilevich AL, Harari PM, Sondel PM. In situ tumor vaccination by combining local radiation and tumor-specific antibody or immunocytokine treatments. Cancer Res 2016; 76 (13): 3929–3941.Morris ZS, Guy EI, Werner LR, Carlson PM, Heinze CM, Kler JS, Busche SM, Jaquish AA, Sriramaneni RN, Carmichael LL, Loibner H, Gillies SD, Korman AJ, Erbe AK, Hank JA, Rakhmilevich AL, Harari PM, Sondel PM. Tumor-specific inhibition of in situ vaccination by distant untreated tumor sites. Cancer Immunol Res 2018; 6(7): 825–834.Ethics ApprovalAll animal work was approved by the University of Wisconsin Institutional Animal Care and Use Committees.</jats:sec

Single cell metabolic imaging of tumor and immune cells in vivo in melanoma bearing mice

IntroductionMetabolic reprogramming of cancer and immune cells occurs during tumorigenesis and has a significant impact on cancer progression. Unfortunately, current techniques to measure tumor and immune cell metabolism require sample destruction and/or cell isolations that remove the spatial context. Two-photon fluorescence lifetime imaging microscopy (FLIM) of the autofluorescent metabolic coenzymes nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) provides in vivo images of cell metabolism at a single cell level.MethodsHere, we report an immunocompetent mCherry reporter mouse model for immune cells that express CD4 either during differentiation or CD4 and/or CD8 in their mature state and perform in vivo imaging of immune and cancer cells within a syngeneic B78 melanoma model. We also report an algorithm for single cell segmentation of mCherry-expressing immune cells within in vivo images.ResultsWe found that immune cells within B78 tumors exhibited decreased FAD mean lifetime and an increased proportion of bound FAD compared to immune cells within spleens. Tumor infiltrating immune cell size also increased compared to immune cells from spleens. These changes are consistent with a shift towards increased activation and proliferation in tumor infiltrating immune cells compared to immune cells from spleens. Tumor infiltrating immune cells exhibited increased FAD mean lifetime and increased protein-bound FAD lifetime compared to B78 tumor cells within the same tumor. Single cell metabolic heterogeneity was observed in both immune and tumor cells in vivo.DiscussionThis approach can be used to monitor single cell metabolic heterogeneity in tumor cells and immune cells to study promising treatments for cancer in the native in vivo context.</jats:sec

598 Local radiation in combination with CpG and anti-OX40 induces enhanced T cell activation and proliferation

BackgroundWe, and others, have previously shown that the in-situ vaccine of hypomethylated CG-enriched oligodeoxynucleotide (CpG) with agonist anti-OX40 antibody (OX40) is effective at curing mice in the A20 lymphoma model [1–4]. In separate preclinical models where CpG+OX40 fails to cause tumor regression, radiation therapy (RT) prior to the in-situ vaccine enhances the anti-tumor effect of CpG+OX40 [4]. We investigated the immune response, and specifically the activity of T cells, following treatment with RT+CpG+OX40 in the B78 melanoma model where CpG+OX40 typically fails to cause tumor regression.MethodsC57BL/6 mice were inoculated with 2x106 B78 melanoma cells on the right flank and allowed to grow until the average tumor size was ~150mm3. In two independent experiments, mice were randomized (n=4–5 per group per experiment) and treated with one of the following: 1) PBS, 2) CpG+OX40, 3) RT, 4) RT+CpG+OX40. 12 Gy external beam RT was dosed to the flank tumor on day 0 and intratumoral CpG (50µg)+OX40 (20 µg) were given on days 5, 7, and 9 after RT. Spleens and tumor draining lymph nodes (TDLNs) were harvested on day 12. T cell activation and proliferation were assessed via flow cytometry.ResultsCompared to all other groups in the study, mice treated with RT+CpG+OX40 demonstrated significantly elevated levels of CD4+ and CD8+ T cell activation in the TDLNs, as measured by interferon gamma expression. Similar trends of CD4+ and CD8+ T cell activation were measured in the spleens. Splenic CD8+ T cells from RT+CpG+OX40 treated mice demonstrated significantly elevated levels of proliferation over PBS and RT, as measured by Ki67.ConclusionsIn B78 melanoma, a weakly immunologic tumor model, combining RT with the in-situ vaccine CpG+OX40 enhances the activity of T cells, evidenced by significantly increased CD4+ and CD8+ T cell activation in the TDLN and spleen and elevated CD8+ T cell proliferation in the spleen.ReferencesHouot, R. and Levy, R. T-cell modulation combined with intratumoral CpG cures lymphoma in a mouse model without the need for chemotherapy. Blood, 2009. 113(15):3546–52.Marabelle, A., et al. Depleting tumor-specific Tregs at a single site eradicates disseminated tumors. J Clin Invest, 2013. 123(6):2447–63.Sagiv-Barfi, I., et al. Eradication of spontaneous malignancy by local immunotherapy. Sci Transl Med, 2018. 10(426).Zangl, LM. Et al. External Beam Radiotherapy Required for Tumor Regression When Using CpG-Oligodeoxynucleotide and Anti-OX40 in an Immunologically Cold Tumor Model. Red Journal. 2019. 105:S88.</jats:sec