In Vivo Vaccination with Cell Line-Derived Whole Tumor Lysates – Neoantigen Quality, Not Quantity Matters

Abstract Background: Cancer vaccines provide a complex source of neoantigens. Still, increasing evidence reveals that the neoantigen quality rather than the quantity is predictive for treatment outcome. Methods: Using the preclinical Mlh1-/- tumor model, we performed a side-by side comparison of two autologous cell-line derived tumor lysates (namely 328 and A7450 T1 M1) harboring different tumor mutational burden (TMB; i.e. ultra-high: 328; moderate-high: A7450 T1 M1). Mice received repetitive prophylactic or therapeutic applications of the vaccine. Tumor incidence, immune responses and tumor microenvironment was examined.Results: Both tumor cell lysates delayed tumor formation in the prophylactic setting, with the A7450 T1 M1 lysate being more effective in decelerating tumor growth than the 328 lysate (median overall survival: 37 vs. 25 weeks). Comparable results were achieved in therapeutic setting and could be traced back to antigen-driven immune stimulation. Reactive T cells isolated from A7450 T1 M1-treated mice recognized autologous Mlh1−/− tumor cells in IFNγ ELISpot, but likewise YAC-1 cells, indicative for stimulation of both arms of the immune system. By deciphering local effects, vaccines shaped the tumor microenvironment differently. While A7450 T1 M1 prophylactically vaccinated tumors harbored low numbers of myeloid-derived suppressor cells (MDSC) and elevated CD8-T cell infiltrates, vaccination with the 328 lysate evoked MDSC infiltration. Similar effects were seen in the therapeutic setting with stable disease induction only upon A7450 T1 M1 vaccination. Untangling individual response profiles revealed strong infiltration with LAG3+ and PD-L1+ immune cells when treatments failed, but almost complete exclusion of checkpoint-expressing lymphocytes in long-term survivors. Conclusions: By applying two tumor cell lysates we demonstrate that neoantigen quality outranks quantity. This should be considered prior to designing cancer vaccine-based combination approaches.

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In vivo vaccination with cell line-derived whole tumor lysates: neoantigen quality, not quantity matters

Journal of Translational Medicine ◽

10.1186/s12967-020-02570-y ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Inken Salewski ◽

Yvonne Saara Gladbach ◽

Steffen Kuntoff ◽

Nina Irmscher ◽

Olga Hahn ◽

...

Keyword(s):

Tumor Microenvironment ◽

Tumor Cell ◽

Cell Line ◽

Suppressor Cells ◽

Tumor Model ◽

Tumor Formation ◽

Individual Response ◽

Cell Lysates ◽

Therapeutic Setting

Abstract Background Cancer vaccines provide a complex source of neoantigens. Still, increasing evidence reveals that the neoantigen quality rather than the quantity is predictive for treatment outcome. Methods Using the preclinical Mlh1−/− tumor model, we performed a side-by side comparison of two autologous cell-line derived tumor lysates (namely 328 and A7450 T1 M1) harboring different tumor mutational burden (TMB; i.e. ultra-high: 328; moderate-high: A7450 T1 M1). Mice received repetitive prophylactic or therapeutic applications of the vaccine. Tumor incidence, immune responses and tumor microenvironment was examined. Results Both tumor cell lysates delayed tumor formation in the prophylactic setting, with the A7450 T1 M1 lysate being more effective in decelerating tumor growth than the 328 lysate (median overall survival: 37 vs. 25 weeks). Comparable results were achieved in therapeutic setting and could be traced back to antigen-driven immune stimulation. Reactive T cells isolated from A7450 T1 M1-treated mice recognized autologous Mlh1−/− tumor cells in IFNγ ELISpot, but likewise YAC-1 cells, indicative for stimulation of both arms of the immune system. By deciphering local effects, vaccines shaped the tumor microenvironment differently. While A7450 T1 M1 prophylactically vaccinated tumors harbored low numbers of myeloid-derived suppressor cells (MDSC) and elevated CD8-T cell infiltrates, vaccination with the 328 lysate evoked MDSC infiltration. Similar effects were seen in the therapeutic setting with stable disease induction only upon A7450 T1 M1 vaccination. Untangling individual response profiles revealed strong infiltration with LAG3+ and PD-L1+ immune cells when treatments failed, but almost complete exclusion of checkpoint-expressing lymphocytes in long-term survivors. Conclusions By applying two tumor cell lysates we demonstrate that neoantigen quality outranks quantity. This should be considered prior to designing cancer vaccine-based combination approaches.

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In vivo vaccination with cell line-derived whole tumor lysates – neoantigen quality, not quantity matters

10.21203/rs.3.rs-51223/v2 ◽

2020 ◽

Author(s):

Inken Salewski ◽

Yvonne Saara Gladbach ◽

Steffen Kuntoff ◽

Nina Irmscher ◽

Olga Hahn ◽

...

Keyword(s):

Tumor Microenvironment ◽

Tumor Cell ◽

Cell Line ◽

Suppressor Cells ◽

Tumor Model ◽

Tumor Formation ◽

Individual Response ◽

Cell Lysates ◽

Therapeutic Setting

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Overexpression of PRDX4 Modulates Tumor Microenvironment and Promotes Urethane-Induced Lung Tumorigenesis

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/8262730 ◽

2020 ◽

Vol 2020 ◽

pp. 1-11

Author(s):

Jianbo Zheng ◽

Xin Guo ◽

Yuka Nakamura ◽

Xiaolei Zhou ◽

Reimon Yamaguchi ◽

...

Keyword(s):

Oxidative Stress ◽

Lung Cancer ◽

Tumor Microenvironment ◽

Tumor Cell ◽

Lung Tumor ◽

Interleukin 1 ◽

Tumor Development ◽

Tumor Model ◽

Tumor Formation ◽

Tumor Diameter

Peroxiredoxin 4 (PRDX4), initially reported as an antioxidant, is overexpressed in lung cancer and participates in its progression. However, its role in the urethane-induced lung tumor model is undetermined. The aim of this study was to investigate the effect of PRDX4 overexpression on carcinogen-induced lung tumor development. Human PRDX4 overexpression transgenic (Tg) mice (hPRDX4+/+) and non-Tg mice were intraperitoneally injected with urethane to induce lung tumor. After 6 months, tumor formation was compared between groups and possible mechanisms for the difference in tumor development were investigated. The serum and lung PRDX4 expressions were enhanced after urethane stimulation in Tg mice. Both the average number of tumors (≥0.5 mm) and tumor diameter per mouse in the Tg group were significantly larger than in non-Tg controls, while body weight was lower in the Tg group. Compared with non-Tg controls, tumor cell proliferation was enhanced, while tumor cell apoptosis was suppressed in Tg mice. Systemic oxidative stress and oxidative stress in lung tumors were inhibited by PRDX4 overexpression. The balance of prooxidant enzymes and antioxidant enzymes was also shifted to a decreased level in Tg tumor. In lung tumor tissue, the density of microvessel penetrated into tumor was higher in the Tg group; macrophage infiltration was enhanced in Tg tumors, while there was no difference in T lymphocyte infiltration; the expressions of cytokines, including interleukin-1 beta (IL-1β) and matrix metallopeptidase 9 (MMP9), were elevated in Tg tumors, which resulted from enhanced phosphorylation of nuclear factor-κB p65 (NF-κB p65) and c-Jun, respectively. In conclusion, PRDX4 overexpression modulated tumor microenvironment and promoted tumor development in the mouse urethane-induced lung cancer model.

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Suppression of tumorigenicity in Wilms tumor by the p15.5-p14 region of chromosome 11

Science ◽

10.1126/science.254.5029.293 ◽

1991 ◽

Vol 254 (5029) ◽

pp. 293-295

Author(s):

SF Dowdy ◽

CL Fasching ◽

D Araujo ◽

KM Lai ◽

E Livanos ◽

...

Keyword(s):

Tumor Cell ◽

Cell Line ◽

Nude Mice ◽

Wilms Tumor ◽

Genetic Locus ◽

Tumor Cell Line ◽

Tumor Formation ◽

The Other ◽

Chromosome 11

Wilms tumor has been associated with genomic alterations at both the 11p13 and 11p15 regions. To differentiate between the involvement of these two loci, a chromosome 11 was constructed that had one or the other region deleted, and this chromosome was introduced into the tumorigenic Wilms tumor cell line G401. When assayed for tumor-forming activity in nude mice, the 11p13-deleted, but not the 11p15.5-p14.1-deleted chromosome, retained its ability to suppress tumor formation. These results provide in vivo functional evidence for the existence of a second genetic locus (WT2) involved in suppressing the tumorigenic phenotype of Wilms tumor.

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TIGIT/CD155 axis mediates resistance to immunotherapy in patients with melanoma with the inflamed tumor microenvironment

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-003134 ◽

2021 ◽

Vol 9 (11) ◽

pp. e003134

Author(s):

Shusuke Kawashima ◽

Takashi Inozume ◽

Masahito Kawazu ◽

Toshihide Ueno ◽

Joji Nagasaki ◽

...

Keyword(s):

T Cells ◽

Tumor Microenvironment ◽

Tumor Cell ◽

Cell Line ◽

Acquired Resistance ◽

Tumor Cell Line ◽

Clinical Samples ◽

Tumor Cell Lines ◽

Patients With Cancer

BackgroundPatients with cancer benefit from treatment with immune checkpoint inhibitors (ICIs), and those with an inflamed tumor microenvironment (TME) and/or high tumor mutation burden (TMB), particularly, tend to respond to ICIs; however, some patients fail, whereas others acquire resistance after initial response despite the inflamed TME and/or high TMB. We assessed the detailed biological mechanisms of resistance to ICIs such as programmed death 1 and/or cytotoxic T-lymphocyte-associated protein 4 blockade therapies using clinical samples.MethodsWe established four pairs of autologous tumor cell lines and tumor-infiltrating lymphocytes (TILs) from patients with melanoma treated with ICIs. These tumor cell lines and TILs were subjected to comprehensive analyses and in vitro functional assays. We assessed tumor volume and TILs in vivo mouse models to validate identified mechanism. Furthermore, we analyzed additional clinical samples from another large melanoma cohort.ResultsTwo patients were super-responders, and the others acquired resistance: the first patient had a non-inflamed TME and acquired resistance due to the loss of the beta-2 microglobulin gene, and the other acquired resistance despite having inflamed TME and extremely high TMB which are reportedly predictive biomarkers. Tumor cell line and paired TIL analyses showed high CD155, TIGIT ligand, and TIGIT expression in the tumor cell line and tumor-infiltrating T cells, respectively. TIGIT blockade or CD155-deletion activated T cells in a functional assay using an autologous cell line and paired TILs from this patient. CD155 expression increased in surviving tumor cells after coculturing with TILs from a responder, which suppressed TIGIT+ T-cell activation. Consistently, TIGIT blockade or CD155-deletion could aid in overcoming resistance to ICIs in vivo mouse models. In clinical samples, CD155 was related to resistance to ICIs in patients with melanoma with an inflamed TME, including both primary and acquired resistance.ConclusionsThe TIGIT/CD155 axis mediates resistance to ICIs in patients with melanoma with an inflamed TME, promoting the development of TIGIT blockade therapies in such patients with cancer.

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The critical immunosuppressive effect of MDSC-derived exosomes in the tumor microenvironment

10.1101/2020.03.05.979195 ◽

2020 ◽

Cited By ~ 3

Author(s):

Mohammad H. Rashid ◽

Thaiz F. Borin ◽

Roxan Ara ◽

Raziye Piranlioglu ◽

Bhagelu R. Achyut ◽

...

Keyword(s):

T Cells ◽

Tumor Microenvironment ◽

Tumor Growth ◽

Cd8 T Cells ◽

Suppressor Cells ◽

Cell Types ◽

Biological Macromolecules

AbstractMyeloid-derived suppressor cells (MDSCs) are an indispensable component of the tumor microenvironment (TME), and our perception regarding the role of MDSCs in tumor promotion is attaining extra layer of intricacy in every study. In conjunction with MDSC’s immunosuppressive and anti-tumor immunity, they candidly facilitate tumor growth, differentiation, and metastasis in several ways that yet to be explored. Alike any other cell types, MDSCs also release a tremendous amount of exosomes or nanovesicles of endosomal origin and partake in intercellular communications by dispatching biological macromolecules. There has not been any experimental study done to characterize the role of MDSCs derived exosomes (MDSC exo) in the modulation of TME. In this study, we isolated MDSC exo and demonstrated that they carry a significant amount of proteins that play an indispensable role in tumor growth, invasion, angiogenesis, and immunomodulation. We observed higher yield and more substantial immunosuppressive potential of exosomes isolated from MDSCs in the primary tumor area than those are in the spleen or bone marrow. Our in vitro data suggest that MDSC exo are capable of hyper activating or exhausting CD8 T-cells and induce reactive oxygen species production that elicits activation-induced cell death. We confirmed the depletion of CD8 T-cells in vivo by treating the mice with MDSC exo. We also observed a reduction in pro-inflammatory M1-macrophages in the spleen of those animals. Our results indicate that immunosuppressive and tumor-promoting functions of MDSC are also implemented by MDSC-derived exosomes which would open up a new avenue of MDSC research and MDSC-targeted therapy.

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Adoptive Transfer of Nf1+/− Bone Marrow Is Sufficient for Neurofibroma Progression in Krox20;Nf1flox/flox Mice.

Blood ◽

10.1182/blood.v106.11.3064.3064 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3064-3064

Author(s):

Fengchun Yang

Keyword(s):

Bone Marrow ◽

Mast Cells ◽

Tumor Microenvironment ◽

Schwann Cells ◽

Genetic Disorder ◽

Hematopoietic Cells ◽

Tumor Formation ◽

Conditional Knockout ◽

Axial Tomography

Abstract Mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), a GTPase activating protein for Ras called neurofibromin. NF1 is a genetic disorder that affects approximately 250,000 individuals in the US, Europe, and Japan alone. Neurofibromas, the hallmark of NF1, are complex tumors characterized by tumorigenic Schwann cells, neoangiogenesis, fibrosis, and degranulating mast cells. Studies in experimental models have emphasized the role of inflammatory cells in altering the microenvironment and facilitating malignant outgrowth. Similarly, Parada (Science, 2002) found that nullizygosity of Nf1 in Schwann cells of conditional knockout mice (Krox20;Nf1flox/flox) was necessary but not sufficient for neurofibroma formation and haploinsufficiency of Nf1 in lineages within the tumor microenvironment was required for neurofibroma progression. We previously provided the first genetic, cellular, and biochemical evidence that haploinsufficiency of Nf1 alters Ras activity and cell fates in mast cells (JEM, 2000, 2001) and identified a mechanism underlying the recruitment of mast cells to tumorigenic Schwann cells (JCI 2003). However, it remains unclear whether Nf1 +/− bone marrow derived hematopoietic cells can directly contribute to neurofibroma formation in vivo. To address this question, Nf1+/− or wildtype (WT) EGFP+ bone marrow (BM) was adoptively transferred into lethally irradiated Krox20;Nf1flox/flox mice and cohorts were followed prospectively for tumor formation using positron emission tomography and computerized axial tomography. Mice transplanted with Nf1+/− but not WT BM developed progressive enlargement of the trigeminal nerve, dorsal root ganglia, peripheral nerves, and motor paralysis similar to Krox20;Nf1flox/− mice that are haploinsufficient at Nf1 in all lineages of the tumor microenvironment. Postmortem analysis revealed that Krox20;Nf1flox/flox mice transplanted with Nf1+/− BM had cellular neurofibromas containing Schwann cells, fibroblasts, blood vessels and mast cells, which closely resembled the cellular architecture of human neurofibromas. Mice transplanted with WT BM did not develop neurofibromas. These studies establish that recruitment of Nf1 +/− BM derived cells to the neurofibroma microenvironment is directly linked to neurofibroma formation and progression. Given our observations, therapies which prevent both the recruitment and the tumor promoting functions of Nf1 +/− hematopoietic cells in neurofibroma formation are currently being tested in vivo as pre-clinical trials.

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IMMU-28. TARGETING IMMUNOSUPPRESSIVE MYELOID DERIVED SUPPRESSOR CELLS VIA MIF/CD74 SIGNALING AXIS TO ATTENUATE GBM GROWTH

Neuro-Oncology ◽

10.1093/neuonc/noz175.521 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi125-vi125

Author(s):

Tyler Alban ◽

Defne Bayik ◽

Balint Otvos ◽

Matthew Grabowski ◽

Manmeet Ahluwalia ◽

...

Keyword(s):

Tumor Microenvironment ◽

Tumor Growth ◽

Immune Cell ◽

Suppressor Cells ◽

Myeloid Derived Suppressor Cells ◽

Signaling Axis ◽

Immunosuppressive Microenvironment ◽

And Function

Abstract The immunosuppressive microenvironment in glioblastoma (GBM) enables persistent tumor growth and evasion from tumoricidal immune cell recognition. Despite a large accumulation of immune cells in the GBM microenvironment, tumor growth continues, and evidence for potent immunosuppression via myeloid derived suppressor cells (MDSCs) is now emerging. In agreement with these observations, we have recently established that increased MDSCs over time correlates with poor prognosis in GBM, making these cells of interest for therapeutic targeting. In seeking to reduce MDSCs in GBM, we previously identified the cytokine macrophage migration inhibitory factor (MIF) as a possible activator of MDSC function in GBM. Here, using a novel in vitro co-culture system to reproducibly and rapidly create GBM-educated MDSCs, we observed that MIF was essential in the generation of MDSCs and that MDSCs generated via this approach express a repertoire of MIF receptors. CD74 was the primary MIF receptor in monocytic MDSCs (M-MDSC), which penetrate the tumor microenvironment in preclinical models and patient samples. A screen of MIF/CD74 interaction inhibitors revealed that MN-166, a clinically relevant blood brain barrier penetrant drug, which is currently fast tracked for FDA approval, reduced MDSC generation and function in vitro. This effect was specific to M-MDSC subsets expressing CD74, and appeared as reduced downstream pERK signaling and MCP-1 secretion. In vivo, MN-166 was able reduce tumor-infiltrating MDSCs, while conferring a significant increase in survival in the syngeneic glioma model GL261. These data provide proof of concept that M-MDSCs can be targeted in the tumor microenvironment via MN-166 to reduce tumor growth and provide a rationale for future clinical assessment of MN-166 to reduce M-MDSCs in the tumor microenvironment. Ongoing studies are assessing the effects of MDSC inhibition in combination with immune activating approaches, in order to inhibit immune suppression while simultaneously activating the immune system.

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Lateral Gene Transfer Contributing to Leukemogenesis

Blood ◽

10.1182/blood.v126.23.1235.1235 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1235-1235

Author(s):

Joo Hyun Lee ◽

Cynthia R. Giver ◽

Sravanti Rangaraju ◽

Edmund K Waller

Keyword(s):

Bone Marrow ◽

Gene Transfer ◽

Tumor Cell ◽

Cell Line ◽

Tumor Cell Line ◽

Hematopoietic Stem ◽

Precursor Frequency ◽

Marrow Cells

Abstract The uncontrolled proliferation of genetically mutated cells is the commonly understood mechanism for cancer growth and invasion, with accumulation of new mutations in daughter cells leading to clonal diversity of cancer derived from a single founding event. The genetic alterations are passed to new generations by cell division and vertical gene transfer. Viral transmission of oncogenes represents a known mechanism of lateral gene transfer in cancer initiation. Some experimental systems have also suggested that circulating DNA or micro-vesicles may contribute to lateral oncogene transfer in tumorigenesis. We hypothesized that interactions between leukemic cells and adjacent normal hematopoietic stem or progenitor cells may provide an alternative mechanism for the accumulation of mutated genes and the multiplicity of distinct clones in leukemia. To test this hypothesis, we performed experiments to determine whether tumorigenic properties could be transferred from a tumor cell line to normal mouse bone marrow cells using both in vivo and in vitro and systems. B6-GFP+ mice were injected i.v. with 200,000 C1498-Luc cells (a B6-derived NKT-cell-like mouse tumor cell line expressing luciferase and DSRed). Bioluminescent imaging was used to monitor the progression of tumor cell growth in recipients. At 1 month after tumor-cell inoculation, marrow from these mice was harvested and FACS-sorted for GFP+ cells (to eliminate C1498 cells), and then cultured on irradiated stromal cell layers in 96-well plates in a limiting dilution analysis for Poisson analysis of GFP+ clonogenic precursor frequency on day 9. On day 10, cells were harvested from culture and GFP+ cells resorted onto fresh stromal layers for second and third determinations of GFP+ clonogenic precursor frequency on days 15 and 18. As shown in Figure 1, the frequency of clonogenic precursors increased with each successive determination for marrow from C1498-injected mice, while control cultures from non-injected mice showed no increase in precursor frequency, suggesting that exposure to C1498 cells conferred a growth advantage to the marrow cells in the tumor-cell injected mice. Similar results were obtained using an in vitro system of co-culture using C1498 cells and GFP+ bone marrow cells, followed by serial rounds of GFP+ sorting and Poisson analysis, showing increases in clonogenic frequency over 5 successive sorts and re-cultures over a 2-month period, while control cultures showed decreased clonogenic frequencies over the course of the experiment. To confirm these observations in vivo, B6-GFP mice were injected with C1498-Luc and marrow was harvested after a month and sorted for GFP+ cells. The sorted marrow was transplanted into 11Gy-irradiated (FVB x B6albino)F1 recipients (5 x 106 cells per recipient, n=5). Control recipients were irradiated and transplanted with GFP+ marrow from non-injected donors. All recipients developed full hematopoietic engraftment with GFP+ cells. At 6 months post-transplant, a tumor was observed near the left shoulder of one of the recipients of C1498-exposed GFP+ marrow. Figure 2 shows IVIS GFP imaging of this mouse with the GFP+ tumor along with control animals. The tumor was not positive for luciferase expression. The mouse was sacrificed and the tumor excised and a portion was dissociated for flow cytometric analysis and culturing (with other segments reserved for subsequent histological and genetic analysis). Both GFP+ and non-GFP cells were found in the dissociated tumor cell suspension. The GFP+ cells were hematopoietic in origin (CD45+) and exhibited a mixed phenotype containing markers expressed on C1498 (DX5+) and myeloid lineage cells (CD11b+) as well as Sca-1, a stem cell marker. Cultures of the GFP+ tumor yielded a population of GFP+ mononuclear cells. These data are consistent with a model in which growth-promoting or transforming genes from cancer cells become incorporated within a healthy hematopoietic stem or progenitor cell, which contributes to the genetic diversity of the cancer through the initiation a new transformed clone. Genetic analysis with deep sequencing will compare the DNA sequences between the parental C1498 cell line, sorted populations of clonogenic GFP+ cells obtained from the in vitro and in vivo experiments, and the GFP+ tumor cells to confirm the transformation of healthy bone marrow hematopoietic stem cells with genetic sequences derived from the C1498 cells. Disclosures No relevant conflicts of interest to declare.

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