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Supplemental Figure 5: Anti-G-CSF treatment restores kidney vascular function in MMTV-PyMT mice.

Supplemental Figure 1: Neutrophils are effectively depleted after treatment with anti-Gr1 targeting antibodies.

Supplemental Figure 2: Neutrophil depletion restores vascular function in kidneys from RIP1-Tag2 mice.

Supplemental figure and table legends

Supplemental Figure 4: Mice with tumors lacking G-CSF expression do not show impaired function of the kidney vasculature.

Supplemental Table 1: Primers used for q-PCR analysis.

Supplemental Figure 3: DNase treatment restores vascular function in kidneys from RIP1-Tag2 mice.

Galectin-1 (Gal1) is a glycan-binding protein that promotes tumor progression by several distinct mechanisms. Through direct binding to vascular endothelial growth factor (VEGF)-receptor 2, Gal1 is able to induce VEGF-like signaling, which contributes to tumor angiogenesis. Furthermore, several studies have demonstrated an immunosuppressive function of Gal1 through effects on both effector and regulatory T cells. Elevated Gal1 expression and secretion have been shown in many tumor types, and high Gal1 serum levels have been connected to poor prognosis in cancer patients. These findings suggest that therapeutic strategies directed against Gal1 would enable simultaneous targeting of angiogenesis, immune evasion and metastasis. In the current study, we have analyzed the potential of Gal1 as a cancer vaccine target. We show that it is possible to generate high anti-Gal1 antibody levels in mice immunized with a recombinant vaccine protein consisting of bacterial sequences fused to Gal1. Growth of Gal1 expressing melanomas was significantly impaired in the immunized mice compared to the control group. This was associated with improved perfusion of the tumor vasculature, as well as increased infiltration of macrophages and cytotoxic T cells (CTLs). The level of granzyme B, mainly originating from CTLs in our model, was significantly elevated in Gal1 vaccinated mice and correlated with a decrease in tumor burden. We conclude that vaccination against Gal1 is a promising pro-immunogenic approach for cancer therapy that could potentially enhance the effect of other immunotherapeutic strategies due to its ability to promote CTL influx in tumors. Authors in thesis list of papers: Julia Femel, Luuk Van Hooren, Falk Saupe, Elisabeth JM Huijbers, Danielle RJ Verboogen, Matthias Reichel, Jessica Cedervall, Victor L Thijssen, Lars Hellman, Arjan W Griffioen, Anna Dimberg, Anna-Karin Olsson

Antigen-specific CD8+T cell accumulation in tumors is a prerequisite for effective immunotherapy, and yet, the mechanisms of lymphocyte transit remain poorly defined. We find that tumor-associated lymphatic vessels control T cell exit from tumors via the chemokine CXCL12, and intratumoral antigen encounter tunes CXCR4 expression on effector CD8+T cells. Only high affinity antigen downregulates CXCR4 and upregulates the CXCL12 decoy receptor, ACKR3, thereby reducing CXCL12 sensitivity and promoting T cell retention. A diverse repertoire of functional tumor-specific CD8+T cells exit the tumor, thereby limiting tumor control. CXCR4 inhibition and loss of lymphatic-specific CXCL12 boosts T cell retention and enhances response to therapeutic immune checkpoint blockade. Strategies that limit T cell egress, therefore, provide a new tool to boost immunotherapy response.One-Sentence SummaryLymphatic vessel-mediated, antigen-dependent CD8+T cell egress limits T cell accumulation in melanomas and impairs anti-tumor immunity.

With the advent of immunotherapies such as immune checkpoint inhibitors and CAR T cells, the interest in the tumor microenvironment and tumor-infiltrating lymphocytes (TILs) cells has increased in recent years as these play a critical role in predicting response to therapy and clinical outcomes. We have reported on the identification of potential targets for CAR T cell therapies by analyzing a multitude of tumor samples with hundreds of antibodies by flow cytometry and a new spatial proteomics platform, the MACSima Imaging Platform (Schäfer et al. Nat. Commun., 2021; Kinkhabwala et.al. Sci. Rep., 2022). Here we report on the analysis of tumor microenvironment and tumor infiltrating immune cells using a novel combined spatial transcriptomics and proteomics approach. For protein expression analysis we generated a panel of 60 fluorochrome conjugated antibodies (REAscreen Immuno-oncology, human, FFPE, version 01) selected to identify immune cells, cancer associated fibroblasts, matrix, blood vessels, lymphatics and malignant epithelial cell populations. RNA analysis was based on a novel in situ hybridization and amplification method followed by a cyclic spatial decoding of transcripts (RNAsky). To standardize and further ease the process of protein and RNA detection we generated the antigen and RNA detection probes in a ready-to-use format, i.e., dried and sealed in 96 well plates ready to be inserted into the MACSima. FFPE specimens of different tumor entities reviewed by a pathologist were first hybridized to probes, which were amplified, and spatially decoded by fluorochrome conjugated oligonucleotides. Subsequently the same specimen was exposed to fluorescently labeled antibodies by cycles of antibody reaction, image acquisition and erasure of signal. Finally, specimens were stained with H&E to report on tissue integrity and correlate spatial orientation. The 2D image stacks were analyzed for transcript and antigen quantification and pattern recognition using both, pixel and segmented single-cell data (MACS iQ View Analysis Software). Across the specimens we identified more than 20 cell types including 12 immune cell subsets. The comparison of the tumor microenvironment, tumor-infiltrating immune cells (TILs) and their spatial organization in relation to the tumor cells allowed to identify differences and similarities across the tumor types. In summary we report here on a novel standardized and automated combined spatial RNA and protein expression analysis of the tumor microenvironment and tumor infiltrating immune cells across tumor types. The characterization dataset demonstrates the ability of our platform to perform standardized in-depth phenotyping of sample cohorts. This will enable discovery and further development of predictive and prognostic biomarkers critical to patient stratification for immunotherapy. Citation Format: Julia Femel, Emily Neil, Dongju Park, Fabio El Yassouri, Anijutta Appelshoffer, Erica Lloyd, Michael DiBuono, Henry Sauer, Hanna Lafayette, Hsinyi Smith, Jinling Wang, Dominic Mangiardi, Alex Makrigiorgos, Paurush Praveen, Silvia Rüberg, Werner Müller, Tanya Wantenaar, Robert Pinard, Andreas Bosio. Analysis of the immune microenvironment and tumor-infiltrating immune cells across different solid tumors by combined spatial transcriptomics and proteomics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5633.

PurposeQuantitative, multiplexed imaging is revealing complex spatial relationships between phenotypically diverse tumor infiltrating leukocyte populations and their prognostic implications. The underlying mechanisms and tissue structures that determine leukocyte distribution within and around tumor nests, however, remain poorly understood. While presumed players in metastatic dissemination, new preclinical data demonstrates that blood and lymphatic vessels (lymphovasculature) also dictate leukocyte trafficking within tumor microenvironments and thereby impact anti-tumor immunity. Here we interrogate these relationships in primary human cutaneous melanoma.Experimental DesignWe established a quantitative, multiplexed imaging platform to simultaneously detect immune infiltrates and tumor-associated vessels in formalin-fixed paraffin embedded patient samples. We performed a discovery, retrospective analysis of 28 treatment-naïve, primary cutaneous melanomas.ResultsHere we find that the lymphvasculature and immune infiltrate is heterogenous across patients in treatment naïve, primary melanoma. We categorized five lymphovascular subtypes that differ by functionality and morphology and mapped their localization in and around primary tumors. Interestingly, the localization of specific vessel subtypes, but not overall vessel density, significantly associated with the presence of lymphoid aggregates, regional progression, and intratumoral T cell infiltrates.ConclusionsWe describe a quantitative platform to enable simultaneous lymphovascular and immune infiltrate analysis and map their spatial relationships in primary melanoma. Our data indicate that tumor-associated vessels exist in different states and that their localization may determine potential for tumor cell exit (metastasis) or leukocyte trafficking (immune response).This platform will support future efforts to map tumor-associated lymphovascular evolution across stage, assess its prognostic value, and stratify patients for adjuvant therapy.TRANSLATIONAL RELEVANCEThis report describes a quantitative, image-based method to investigate the relationship between the tumor-associated lymphovasculature and immune landscape in treatment naïve, primary human melanoma. The research shows that melanoma-associated blood and lymphatic vessels display context-dependent phenotypes that associate both with the risk of regional progression and immune infiltration. These findings indicate that stromal/vascular heterogeneity may underlie regional differences in immunogenicity and thus present opportunities for future biomarker development and therapeutic intervention.

Therapy resistance arises from heterogeneous drug-tolerant persister cells or minimal residual disease (MRD) through genetic and nongenetic mechanisms. A key question is whether specific molecular features of the MRD ecosystem determine which of these two distinct trajectories will eventually prevail. We show that, in melanoma exposed to mitogen-activated protein kinase therapeutics, emergence of a transient neural crest stem cell (NCSC) population in MRD concurs with the development of nongenetic resistance. This increase relies on a glial cell line-derived neurotrophic factor-dependent signaling cascade, which activates the AKT survival pathway in a focal adhesion kinase (FAK)-dependent manner. Ablation of the NCSC population through FAK inhibition delays relapse in patient-derived tumor xenografts. Strikingly, all tumors that ultimately escape this treatment exhibit resistance-conferring genetic alterations and increased sensitivity to extracellular signal-regulated kinase inhibition. These findings identify an approach that abrogates the nongenetic resistance trajectory in melanoma and demonstrate that the cellular composition of MRD deterministically imposes distinct drug resistance evolutionary paths. ispartof: CANCER CELL vol:39 issue:8 pages:1135-+ ispartof: location:United States status: published

SummaryThe ability to predict the future behaviour of an individual cancer is crucial for precision cancer medicine and, in particular, for the development of strategies that prevent acquisition of resistance to anti-cancer drugs. Therapy resistance, which often develops from a heterogeneous pool of drug-tolerant cells known as minimal residual disease (MRD), is thought to mainly occur through acquisition of genetic alterations. Increasing evidence, however, indicates that drug resistance might also be acquired though nongenetic mechanisms. A key emerging question is therefore whether specific molecular and/or cellular features of the MRD ecosystem determine which of these two distinct resistance trajectories will eventually prevail. We show herein that, in melanoma exposed to MAPK-therapeutics, the presence of a neural crest stem cell (NCSC) subpopulation in MRD concurred with the rapid development of resistance through nongenetic mechanisms. Emergence of this drug-tolerant population in MRD relies on a GDNF-dependent autocrine and paracrine signalling cascade, which activates the AKT survival pathway in a Focal-adhesion kinase-(FAK) dependent manner. Ablation of this subpopulation through inhibition of FAK/SRC-signalling delayed relapse in patient-derived tumour xenografts. Strikingly, all tumours that eventually escaped this treatment exhibited resistance-conferring genetic alterations and increased sensitivity to ERK-inhibition. These findings firmly establish that nongenetic reprogramming events contribute to therapy resistance in melanoma and identify a clinically-compatible approach that abrogates such a trajectory. Importantly, these data demonstrate that the cellular composition of MRD deterministically imposes distinct drug resistance evolutionary paths and highlight key principles that may permit more effective pre-emptive therapeutic interventions.

Mechanisms of immune suppression in peripheral tissues counteract protective immunity to prevent immunopathology and are coopted by tumors for immune evasion. While lymphatic vessels facilitate T cell priming, they also exert immune suppressive effects in lymph nodes at steady-state. Therefore, we hypothesized that peripheral lymphatic vessels acquire suppressive mechanisms to limit local effector CD8+ T cell accumulation in murine skin. We demonstrate that nonhematopoietic PD-L1 is largely expressed by lymphatic and blood endothelial cells and limits CD8+ T cell accumulation in tumor microenvironments. IFNγ produced by tissue-infiltrating, antigen-specific CD8+ T cells, which are in close proximity to tumor-associated lymphatic vessels, is sufficient to induce lymphatic vessel PD-L1 expression. Disruption of IFNγ-dependent crosstalk through lymphatic-specific loss of IFNγR boosts T cell accumulation in infected and malignant skin leading to increased viral pathology and tumor control, respectively. Consequently, we identify IFNγR as an immunological switch in lymphatic vessels that balances protective immunity and immunopathology leading to adaptive immune resistance in melanoma.

With the aim to improve the efficacy of therapeutic vaccines that target self-antigens, we have developed a novel fusion protein vaccine on the basis of the C-terminal multimerizing end of the variable lymphocyte receptor B (VLRB), the Ig equivalent in jawless fishes. Recombinant vaccines were produced in Escherichia coli by fusing the VLRB sequence to 4 different cancer-associated target molecules. The anti-self-immune response generated in mice that were vaccinated with VLRB vaccines was compared with the response in mice that received vaccines that contained bacterial thioredoxin (TRX), previously identified as an efficient carrier. The anti-self-Abs were analyzed with respect to titers, binding properties, and duration of response. VLRB-vaccinated mice displayed a 2- to 10-fold increase in anti-self-Ab titers and a substantial decrease in Abs against the foreign part of the fusion protein compared with the response in TRX-vaccinated mice (P < 0.01). VLRB-generated Ab response had duration similar to the corresponding TRX-generated Abs, but displayed a higher diversity in binding characteristics. Of importance, VLRB vaccines could sustain an immune response against several targets simultaneously. VLRB vaccines fulfill several key criteria for an efficient therapeutic vaccine that targets self-antigens as a result of its small size, its multimerizing capacity, and nonexposed foreign sequences in the fusion protein.-Saupe, F., Reichel, M., Huijbers, E. J. M., Femel, J., Markgren, P.-O., Andersson, C. E., Deindl, S., Danielson, U. H., Hellman, L. T., Olsson, A.-K. Development of a novel therapeutic vaccine carrier that sustains high antibody titers against several targets simultaneously.

Cancer produces a variety of collateral effects in patients beyond the malignancy itself, including threats to distal organ functions. However, the basis for such effects, associated with either primary or metastatic tumors, are generally poorly understood. In this study, we show how heart and kidney vascular function is impaired by neutrophils that accumulate in those tissues as a result of tumor formation in two different transgenic mouse models of cancer (RIP1-Tag2 model of insulinoma and MMTV-PyMT model of breast cancer). Neutrophil depletion by systemic administration of an anti-Gr1 antibody improved vascular perfusion and prevented vascular leakage in kidney vessels. We also observed the accumulation of platelet–neutrophil complexes, a signature of neutrophil extracellular traps (NET), in the kidneys of tumor-bearing mice that were completely absent from healthy nontumor-bearing littermates. NET accumulation in the vasculature was associated with upregulation of the proinflammatory adhesion molecules ICAM-1, VCAM-1, and E-selectin, as well as the proinflammatory cytokines IL1β, IL6, and the chemokine CXCL1. Administering DNase I to dissolve NETs, which have a high DNA content, restored perfusion in the kidney and heart to levels seen in nontumor-bearing mice, and also prevented vessel leakage in the blood vasculature of these organs. Taken together, our findings strongly suggest that NETs mediate the negative collateral effects of tumors on distal organs, acting to impair vascular function, and to heighten inflammation at these sites. Cancer Res; 75(13); 2653–62. ©2015 AACR.

Therapeutic vaccination targeting self-molecules is an attractive alternative to monoclonal antibody-based therapies for cancer and various inflammatory diseases. However, development of cancer vaccines targeting self-molecules has proven difficult. One complicating factor is that tumor cells have developed strategies to escape recognition by the immune system. Antigens specifically expressed by the tumor vasculature can therefore provide alternative targets. The alternatively spliced extra domain-A and B (ED-A and ED-B) of fibronectin are expressed during vasculogenesis in the embryo, but essentially undetectable under normal conditions in the adult. However, these domains are re-expressed during tumor angiogenesis and matrix remodeling, which renders them highly interesting for targeted cancer therapies. Using the MMTV-PyMT transgenic model of metastatic mammary carcinoma, we show that tumor burden can be significantly decreased by immunization against ED-A in a therapeutic setting. Furthermore, we found that in mice carrying anti-ED-A antibodies the number of metastases was reduced. ED-A immunization increased infiltration of macrophages and compromised tumor blood vessel function. These findings implicate an attack of the tumor vasculature by the immune system, through a polyclonal antibody response. We conclude that tumor vascular antigens are promising candidates for development of therapeutic vaccines targeting growth of primary tumors as well as disseminated disease.

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

We have recently shown that immunization against the extra domain-B (ED-B) of fibronectin, using Freund's adjuvant, reduces tumor growth in mice by 70%. In the present study we compare the immune response generated against ED-B using the non-toxic and biodegradable adjuvant Montanide ISA 720/CpG with the response elicited by Freund's adjuvant. Montanide ISA 720/CpG induced anti-ED-B antibodies with higher avidity and less variable levels between individuals than Freund's. Moreover, the duration of the immune response was longer and the generation of anti-ED-B antibodies in naïve mice was faster, when Montanide ISA 720/CpG was used. We conclude that it is possible to replace the mineral oil based adjuvant Freund's with an adjuvant acceptable for human use, which is a prerequisite for transfer of the ED-B vaccine to the clinic.

Lymphatic vessel remodeling and lymphangiogenesis is correlated with melanoma progression and lymph node metastasis. While lymphatic vessels provide an important route for disseminating tumor cells, they are also a crucial interface between a developing malignancy and the host immune response. Rather than acting as passive conduits, lymphatic vessels directly regulate their transport function and facilitate leukocyte trafficking for efficient induction of adaptive immunity in downstream draining lymph nodes. We recently published that in the absence of dermal lymphatic vessels, the tumor microenvironment of murine melanoma remains completely uninflamed and fails to induce a robust T-cell response (1). Consistently, TCGA analysis of human cutaneous metastatic melanoma identified positive correlations between LEC gene expression and immune genes, including a T cell-inflamed signature, indicating a relationship between lymphatic vessel remodeling and local immunity. Furthermore, others have recently demonstrated that lymphatic vessel density (LVD) in combination with intratumoral T cell function stratified best overall survival in nonmetastatic and metastatic colorectal cancer (2). In contrast to this, however, many reports independently correlate peritumoral LVD with lymph node metastasis and some poor prognosis (3). Furthermore, our previous work demonstrated that vascular endothelial growth factor C (VEGFC)-driven lymphangiogenesis in the context of murine melanoma drove increased leukocyte infiltration but associated with poor CD8+ T cell priming in draining lymph nodes (4). We therefore hypothesize that lymphatic vessels are (A) required for induction of adaptive immunity but (B) acquire immunosuppressive activity as a function of the accumulation of local cytotoxic immunity. Furthermore, we predict that LVD may be a relevant biomarker of in situ immune responsiveness and response to therapy (5). To test the first part of this hypothesis (A), we have continued our published work and used a cutaneous model of viral infection to demonstrate the requirement for lymphatic vessel drainage in cutaneous immunity. Following cutaneous vaccinia infection we demonstrate that in the absence of lymphatic vessel transport, both cellular and humoral adaptive immune responses fail to initiate, leading to enhanced cutaneous immunopathology and persistent viral load. The complete absence of primed CD8+ and CD4+ T cells in cutaneous tissue following challenge mirrors our observations in melanoma and is consistent with correlations of intratumoral lymphocytes and LVD both by our group in cutaneous metastatic melanoma as well as by others. This unequivocal requirement for a functional lymphatic vasculature in the priming of cutaneous immunity further supports the prediction that LVD may be a novel biomarker of immune reactivity within tumor parenchyma. To test this, we simultaneously evaluated immune and vascular components in human primary melanoma samples using a multiplex-immunohistochemistry-based approach. Tissue regions that include tumor/stroma borders and show high CD8+ T-cell infiltrates are selected for analysis, followed by tissue segmentation, and automated detection of cell populations within intratumoral regions and bordering stroma. Interestingly, those tumors with enhanced hematopoietic infiltrate (CD68 and CD8) also appear to demonstrate increased vasculature, both blood (CD31 and CD34) and lymphatic (D2-40 and Prox1). Preliminary data demonstrate that lymphatic vessels, blood vessels, and CD8+ T cells are significantly enriched at the tumor-stroma border in primary melanoma and positively correlate with one another, indicating that lymphatic vessels may be a dynamic component of the “T cell-inflamed” microenvironment. While we demonstrate that lymphatic vessels are necessary for immune induction, we further hypothesized (B) that in the context of an ongoing immune response lymphatic vessels adapt their function to promote immune resolution. The adaptive resistance hypothesis proposes that upon accumulation of local cytotoxic immunity, both tumors as well as stromal components adapt and acquire therapeutically relevant immunosuppressive function. We demonstrate that peripheral, tumor-associated lymphatic endothelial cells (LEC; CD45-CD31+gp38+) acquire expression of immunoregulatory proteins, most notably programmed death receptor ligand-1 (PD-L1) and major histocompatibility complex II (MHCII), coincident with CD8+ T-cell infiltration in an interferon gamma (IFNg)-dependent manner. Adoptive transfer of activated CD8+ T cells induced higher expression of PD-L1 by LECs in B16 F10 tumors, while neutralization of IFNg reduced levels to that of naïve skin. Furthermore, conditional knockout of the IFNgR (Lyve1-Cre) prevented upregulation of PD-L1 on tumor-associated LECs. Notably, lymph node LECs constitutively express PD-L1 and this expression contributes to the attenuation of self-reactive CD8+ T-cell responses (6). Importantly, in the absence of IFNgR on peripheral LECs we observed significantly enhanced accumulation of antigen-specific CD8+ T cells in cutaneous tissue. Thus, cutaneous lymphatic vessels, while necessary for immune induction, acquire immunodulatory properties in a context-dependent manner and may participate in immune evasion within tumor microenvironments. In conclusion, our work across multiple model systems provides strong experimental evidence to indicate that the lymphatic vasculature is an important, active component of the antitumor immune response and may represent a biomarker to stratify patient response and survival for effective clinical immunotherapy. These data indicate a need to revisit the passive paradigm of lymphatic vessel involvement in tumor progression and metastasis to a more active model whereby lymphatic vessels are both required for antitumor immunity but functionally evolve in response to accumulating cytotoxicity to drive immune evasion. References: 1. Lund AW et al. Lymphatic vessels regulate immune microenvironments in human and murine melanoma. J Clin Invest 2016;126:3389-402. doi: 10.1172/JCI79434. 2. Mlecnik B et al. The tumor microenvironment and Immunoscore are critical determinants of dissemination to distant metastasis. Sci Transl Med 2016;8:327ra26. 3. Pasquali S et al. Lymphatic biomarkers in primary melanomas as predictors of regional lymph node metastasis and patient outcomes. Pigment Cell Melanoma Res 2013;26:326-37. 4. Lund AW et al. VEGF-C promotes immune tolerance in B16 melanomas and cross-presentation of tumor antigen by lymph node lymphatics. Cell Rep 2012;1:191-9. 5. Lund AW. Rethinking lymphatic vessels and antitumor immunity. Trends Cancer 2016;2:548-51. doi: 10.1016/j.trecan.2016.09.0056. Tewalt EF et al. Lymphatic endothelial cells induce tolerance via PD-L1 and lack of costimulation leading to high-level PD-1 expression on CD8 T cells. Blood 2012;120:4772-82. Citation Format: Ryan S. Lane, Julia Femel, Jamie Booth, Christopher Loo, Nicholas Nelson, Takahiro Tsujikawa, Guillaume Thibault, Amanda W. Lund. Lymphatic vessels: Balancing immune priming and immune evasion in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr NG02. doi:10.1158/1538-7445.AM2017-NG02

The aim of our work is to assess the local lymphatic vasculature within the tumor microenvironment as a possible biomarker for response to immunotherapy. Targeting the immune checkpoint molecules CTLA-4 and PD-1 is showing unprecedented clinical impact in human melanoma patients. However, a significant subset of patients does not respond to therapy. Therefore efforts have been made to identify biomarkers predictive of survival and response to immunotherapy, and evaluation of the immune microenvironment was shown to have high prognostic power. Characterization of tumor-infiltrating T cells with respect to density, phenotype and location has been demonstrated to predict survival and metastasis in retrospective studies and might be superior to classical TNM staging. Despite these promising results current strategies are not 100% predictive and additional cell populations within the tumor microenvironment are likely to affect the overall anti-tumor immune response. Interestingly, the density of tumor-associated lymphatic vessels was found to be decreased in patients with metastatic colorectal cancer compared to non-metastatic patients. In addition, our recent work using metastatic melanoma samples from the Broad Institute's TCGA database demonstrated that expression of lymphatic genes (LYVE-1 and podoplanin) correlates with expression levels of immune cell markers. In mice lacking dermal lymphatic vessels immune infiltration into experimental melanoma was significantly decreased. At the same time these tumors displayed decreased immune suppression and improved tumor control by transferred cytotoxic T cells. These results suggest that lymphatic vessels are essential for establishment of an efficient anti-tumor immune response, but have a role in negatively regulating anti-tumor immunity during tumor progression. We therefore hypothesize that local lymphatic vessel density within the tumor microenvironment predicts immune infiltrate and response to immunotherapy. To simultaneously evaluate immune and vascular components in human formalin-fixed samples of melanoma we are using a multiplex-immunohistochemistry-based approach, which allows for examination of up to twelve markers by sequential staining of a single marker at a time. Tissue regions that include tumor/stroma borders and show high CD8+ T cell infiltrates are selected for analysis. This is followed by tissue segmentation based on the presence of a tumor marker and automated detection of cell populations within intra-tumoral regions and the invasive margin at the tumor border. Analysis using image cytometry allows us to quantify and correlate presence and location of multiple cell types within the tumor and the surrounding stroma. Our work will contribute to improved stratification of cancer patients with respect to possible response to immunotherapy. Citation Format: Julia Femel, Takahiro Tsujikawa, Guillaume Thibault, Jamie Booth, Amanda W. Lund. Lymphatic vessels as a biomarker in human melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A19.

Monoclonal antibody-based therapies have made an important contribution to current treatment strategies for cancer and autoimmune disease. However, the cost for these new drugs puts a significant strain on the health-care economy, resulting in limited availability for patients. Therapeutic vaccination, defined as induction of immunity against a disease-related self-molecule, is therefore an attractive alternative. To analyze the potential of such an approach, we have developed a vaccine against the extra domain-B (ED-B) of fibronectin. This 91-aa domain, inserted by alternative splicing, is expressed during vasculogenesis in the embryo, but essentially undetectable under normal conditions in the adult. However, ED-B is highly expressed around angiogenic vasculature, such as in tumorigenesis. Here, we demonstrate that it is possible to break self-tolerance and induce a strong antibody response against ED-B by vaccination. Nineteen of 20 vaccinated mice responded with production of anti-ED-B antibodies and displayed a 70% reduction in tumor size compared to those lacking anti-ED-B antibodies. Analysis of the tumor tissue revealed that immunization against ED-B induced several changes, consistent with an attack by the immune system. These data show that tumor vascular antigens are highly interesting candidates for development of therapeutic vaccines targeting solid tumors.