Immuno-oncology Research Research Articles

Using Spectral Flow Cytometry to Characterize Anti-Tumor Immunity in Orthotopic and Subcutaneous Mouse Models of Cancer.

Mouse models remain at the forefront of immuno-oncology research, providing invaluable insights into the complex interactions between the immune system and developing tumors. While several flow cytometry panels have been developed to study cancer immunity in mice, most are limited in their capacity to address the complexity of anti-cancer immune responses. For example, many of the panels developed to date focus on a restricted number of leukocyte populations (T cells or antigen-presenting cells), failing to include the multitude of other subsets that participate in anti-cancer immunity. In addition, these panels were developed using blood or splenic leukocytes. While the immune composition of the blood or spleen can provide information on systemic immune responses to cancer, it is in the tumor microenvironment (TME) that local immunity takes place. Therefore, we optimized this spectral flow cytometry panel to identify the chief cell types that take part in cancer immunity using immune cells from cancer tissue. We used pancreatic tumors implanted both orthotopically and subcutaneously to demonstrate the panel's flexibility and suitability in diverse mouse models. The panel was also validated in peripheral immune districts (the blood, spleen, and liver of tumor-bearing mice) to allow comparisons between local and systemic anti-tumor immunity. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tumor induction-Orthotopic Alternate Protocol: Tumor induction-Subcutaneous Basic Protocol 2: Preparation of single-cell suspensions from the tumor, spleen, liver, and blood of tumor-bearing mice Basic Protocol 3: Staining single-cell suspensions from the tumor, spleen, liver, and blood of tumor-bearing mice.

Advancing drug development by leveraging microdissection for proteogenomic analysis of histological sections.

e14660 Background: Combining laser capture microdissection (LCM) with proteogenomics in cancer research offers a targeted way to way to explore the intricate interactions between the tumor microenvironment and tumor cells proper. For immuno-oncology (IO) research, this is especially important, since improvements in the predictability of patient response to IO-based drugs is greatly needed. An improved understanding of the spatial relationships involving tumor cells, stromal cells, blood supply and immune cell interactions may help to inform drug development and improve the indicators of success for IO-based drug regimens. Methods: From fresh frozen tissue, LCM is used to isolate and obtain distinct histological cell types. Nucleic acids and proteins are extracted from captured cells for in-depth multi-modality molecular profiling assays. This includes multiple genomic modalities and a customized solution-based mass spectrometry proteomics approach for samples or specimens with a limited amount of tissue. Results: Optimizing the analysis of minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, DNA sequencing can be performed for the discovery and analysis of actionable mutations, copy number variation, and methylation profiles. RNA-seq may be performed for gene expression. Regarding genomic-based studies, commercial kits are often utilized for these steps due to their general availability, rapid innovation, and the robust performance of these products. However, this kit-based adaptive approach is not true for proteomics. There remains a need for highly sensitive proteomic methods targeting small-sized samples. Our novel proteogenomics approach is focused around a customized enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. This includes an in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Conclusions: A proteogenomic approach is reported that is utilizing amount limited tissue quantities from microdissected fresh frozen tissue. The methodology may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.

Assessing personalized responses to anti-PD-1 treatment using patient-derived lung tumor-on-chip

There is a compelling need for approaches to predict the efficacy of immunotherapy drugs. Tumor-on-chip technology exploits microfluidics to generate 3D cell co-cultures embedded in hydrogels that recapitulate simplified tumor ecosystems. Here, we present the development and validation of lung tumor-on-chip platforms to quickly and precisely measure exvivo the effects of immune checkpoint inhibitors on Tcell-mediated cancer cell death by exploiting the power of live imaging and advanced image analysis algorithms. The integration of autologous immunosuppressive FAP+ cancer-associated fibroblasts impaired the response to anti-PD-1, indicating that tumors-on-chips are capable of recapitulating stroma-dependent mechanisms of immunotherapy resistance. For a small cohort of non-small cell lung cancer patients, we generated personalized tumors-on-chips with their autologous primary cells isolated from fresh tumor samples, and we measured the responses to anti-PD-1 treatment. These results support the power of tumor-on-chip technology in immuno-oncology research and open a path to future clinical validations.

Bridging the Gap from Bench to Bedside: A Call for In Vivo Preclinical Models to Advance Endometrial Cancer and Cervical Cancer Immuno-oncology Research.

Advanced-stage endometrial and cervical cancers are associated with poor outcomes despite contemporary advances in surgical techniques and therapeutics. Recent clinical trial results have led to a shift in the treatment paradigm for both malignancies, in which immunotherapy is now incorporated as the standard of care up front for most patients with advanced endometrial and cervical cancers as the standard of care. Impressive response rates have been observed, but unfortunately, a subset of patients do not benefit from immunotherapy, and survival remains poor. Continued preclinical research and clinical trial development are crucial for our understanding of resistance mechanisms to immunotherapy and maximization of therapeutic efficacy. In this setting, syngeneic models are preferred over xenograft models as they allow for the evaluation of the tumor-immune interaction in an immunocompetent host, most closely mimicking the tumor-immune interaction in patients with cancer. Unfortunately, significant disparities exist about syngeneic models in gynecologic malignancy, in which queries from multiple large bioscience companies confirm no commercial availability of endometrial or cervical cancer syngeneic cell lines. Published data exist about the recent development of several endometrial and cervical cancer syngeneic cell lines, warranting further investigation. Closing the disparity gap for preclinical models in endometrial and cervical cancers will support physician scientists, basic and translational researchers, and clinical trialists who are dedicated to improving outcomes for our patients with advanced disease and poor prognosis.

Single-cell RNA sequencing reveals the cellular and molecular heterogeneity of treatment-naïve primary osteosarcoma in dogs

Osteosarcoma (OS) is a heterogeneous, aggressive malignancy of the bone that disproportionally affects children and adolescents. Therapeutic interventions for OS are limited, which is in part due to the complex tumor microenvironment (TME). As such, we used single-cell RNA sequencing (scRNA-seq) to describe the cellular and molecular composition of the TME in 6 treatment-naïve dogs with spontaneously occurring primary OS. Through analysis of 35,310 cells, we identified 41 transcriptomically distinct cell types including the characterization of follicular helper T cells, mature regulatory dendritic cells (mregDCs), and 8 tumor-associated macrophage (TAM) populations. Cell-cell interaction analysis predicted that mregDCs and TAMs play key roles in modulating T cell mediated immunity. Furthermore, we completed cross-species cell type gene signature homology analysis and found a high degree of similarity between human and canine OS. The data presented here act as a roadmap of canine OS which can be applied to advance translational immuno-oncology research.

Abstract 730: Leveraging microdissection for proteogenomic analysis of histological sections to advance drug development

Abstract Background: Combining proteogenomics with laser capture microdissection (LCM) in cancer research offers a targeted way to explore the intricate interactions between tumor cells and the tumor microenvironment. This is especially important for immuno-oncology (IO) research where improvements in the predictability of patient response to IO-based drugs is sorely needed. An improved understanding of the spatial relationships involving tumor cells proper, stromal cells, blood supply and immune cell interactions may help to inform drug development and improve the indicators of success for IO-based drug regimens. Methods: LCM is used to isolate and obtain distinct histological cell types from fresh frozen tissue. Once cells have been captured, nucleic acids and proteins are extracted for in-depth multi-modality molecular profiling assays involving multiple genomic modalities and a customized solution-based mass spectrometry proteomics approach for samples or specimens with a limited amount of tissue. Results: Optimizing analysis of minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, RNA-seq may be performed for gene expression and DNA sequencing performed for the discovery and analysis of actionable mutations, copy number variation, and methylation profiles. Regarding genomic-based studies, commercial kits are often utilized for these steps due to their general availability, rapid innovation, and the robust performance of these products. However, this kit-based adaptive approach is not true for proteomics. There remains a need for highly sensitive proteomic methods targeting small-sized samples. A significant part of our proteogenomics approach is focused around an enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. This includes an in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Conclusions: Reported here is a proteogenomic approach that is leveraging from microdissected fresh frozen tissue. The methodology may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume. Citation Format: Ik Jae Shin, Michael Tangrea, Michael R. Emmert-Buck, Donald J. Johann. Leveraging microdissection for proteogenomic analysis of histological sections to advance drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 730.

Deciphering a Prognostic Signature Based on Soluble Mediators Defines the Immune Landscape and Predicts Prognosis in HNSCC.

The study on Head and Neck Squamous Cell Carcinoma (HNSCC), a prevalent and aggressive form of head and neck cancer, focuses on the often-overlooked role of soluble mediators. The objective is to leverage a transcriptome-based risk analysis utilizing soluble mediator-related genes (SMRGs) to provide novel insights into prognosis and immunotherapy efficacy in HNSCC patients. We analyzed the expression and prognostic significance of 10,859 SMRGs using 502 HNSCC and 44 normal samples from the TCGA-HNSC cohort in The Cancer Genome Atlas (TCGA). The samples were divided into training and test sets in a 7:3 ratio, with an additional external validation using 40 tumor samples from the International Cancer Genome Consortium (ICGC). Key differentially expressed genes (DEGs) with prognostic significance were identified through univariate and Lasso-Cox regression analyses. A prognostic model based on 20 SMRGs was developed using Lasso and multivariate Cox regression. We assessed the clinical outcomes and immune status in high-risk (HR) and low-risk (LR) HNSCC patients utilizing the BEST databases and single-sample Gene Set Enrichment Analysis (ssGSEA). The 20 SMRGs were crucial in predicting the prognosis of HNSCC, with the SMRG signature emerging as an independent prognostic indicator. Patients classified in the HR group exhibited poorer outcomes compared to those in the LR group. A nomogram, integrating clinical characteristics and risk scores, demonstrated substantial prognostic value. Immunotherapy appeared to be more effective in the LR group, possibly attributed to enhanced immune infiltration and expression of immune checkpoints. The model based on soluble mediator-associated genes offers a fresh perspective for assessing the pre-immune efficacy and showcases robust predictive capabilities. This innovative approach holds significant promise in advancing the field of precision immuno-oncology research, providing valuable insights for personalized treatment strategies in HNSCC.

Abstract 88: Novel cell-based bioassays enable preclinical animal studies during immuno-oncology drug development

Abstract Translation of immuno-oncology research into novel therapies relies on animal models to advise target selection and portray mechanism of action (MoA) and pharmacokinetics of lead candidates. Mouse, monkey, and more recently, canine models of human cancers are being utilized to this end. However, one major challenge in developing and utilizing these models is the lack of technology to characterize non-human analogs of therapeutic antibodies. Herein, we report the development of novel cell-based bioassays for mouse, monkey, and canine targets to enable immuno-oncology preclinical studies. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are important MoAs for antibody-based cancer immunotherapies. We have developed a suite of cell-based bioassays, each expressing a corresponding animal Fcγ receptor (FcγR) and a luciferase reporter that responds to FcγR activation. These assays exhibit sensitive and quantitative luciferase readouts of FcγR activation. Animal models are further used to provide insight into the potential efficacy of immune checkpoint (IC) inhibitors. Programmed cell death protein 1 (PD-1) is an IC receptor that negatively regulates T cell function. Blockade of PD-1 has demonstrated clinical efficacy, but there remains a significant fraction of patients unresponsive to this therapy. Combination of PD-1 inhibitors with other interventions has been a significant focus for many programs. However, tools for functional validation of non-human PD-1 inhibitors are lacking. We report here the development of two new bioassays for functional characterization of PD-1/PD-L1 inhibitors for mouse and canine targets. Each of these assays consists of two engineered cell lines: a T effector cell line that express a luciferase reporter driven by specific promoter/response elements responding to the intracellular signals mediated by the T cell receptor (TCR), with modulation from PD-1, and an artificial antigen presenting cell line (aAPC). The bioassays are sensitive, quantitative, and have demonstrated characteristics required for potency assays for validation of antibodies in preclinical combination studies. Citation Format: Jun Wang, Denise Garvin, Aileen Paguio, Elizabeth Perrin, Jim Hartnett, Mei Cong, Jamison Grailer. Novel cell-based bioassays enable preclinical animal studies during immuno-oncology drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 88.

Abstract 6181: Digital pathology workflow: Building a bridge for translational biomarker development in humanized mice

Abstract The introduction of immune checkpoint inhibitors (ICIs) directed against programmed death protein 1 (PD-1), as well as cytotoxic T lymphocyte antigen 4 (CTLA-4), has revolutionized the treatment of non-small cell lung cancer (NSCLC) in the last decade. These therapies have shown significant success in a subset of NSCLC patients, leading to durable response and improved overall survival. However, clear predictive biomarkers for response are still yet to be defined. Tumor-infiltrating lymphocytes (TILs) as one important part of the tumor microenvironment (TME) have proven to be predictive biomarkers in other tumor types such as melanoma, whereas not fully understood in NSCLC. The assessment of TILs typically involves techniques such as flow cytometry (FC) or immunohistochemistry (IHC). While the former has the advantage of being quantitative and relatively easy to standardize it lacks translational value as most clinical samples will be examined using IHC. The current study aims to compare both methods at terminal end point and for treatment outcome, using a panel of 14 NSCLC PDX models, implanted in humanized NSG mice and treated with anti-CTLA4, anti-PD1 and the combination thereof. Based on FC analyses of the untreated tumor tissue the PDX models were stratified into seven hot (>5% TILs) and seven cold (<5% TILs) tumors. The IHC samples were analyzed by digital pathology for TIL infiltration by making use of automated segmentation of the three main tissue classes: tumor, stroma and necrosis. Small number of IHC slides were manually annotated to form a training set, which were then used to train a deep learning model using Visiopharm, a commercial AI platform for image analysis. By quantification of TILs in a spatial context of the tumor tissue, across all models, tumor was the most prominent tissue class. The tissue class distribution as well as the TIL rate (FC or IHC measure) is a model immanent feature. Under treatment, the ratio was stable in case of the cold tumors. In the hot tumors the stroma compartment increased while tumor tissue decreased. Treatment sensitive models displayed an increase of TILs confirmed by FC and IHC. The latter revealed that the TILs specifically increased in the tumor tissue whereas decreased or was stable in the stroma. This observation was significantly more pronounced in cold tumors compared to hot tumors (Oneway ANOVA, p < 0.001). Furthermore, a positive correlation between FC and IHC results on CD45 positive cells in the tumors (p< 0.002) could be confirmed. In this study, we proofed the feasibility of software-based image analysis as translational relevant read-out in preclinical animal experiments. The possibility to reproducibly quantify tissue classes, immune cell markers and including spatial information paves the way towards discovery of novel features predicting response in translational immuno-oncology research. Citation Format: Matti Hakkarainen, Timmo Bragge, Jussi Rytkönen, Eva Oswald, Julia B. Schueler. Digital pathology workflow: Building a bridge for translational biomarker development in humanized mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6181.

Abstract 4240: A comprehensive in vitro tumor model: One-stop simultaneous isolation and culture of tumoroids, CAFs and TILs for advancing immuno-oncology research

Abstract The tumor microenvironment (TME), composed of a diverse array of cells, jointly participates in tumor formation and has attracted attention as a direct or indirect therapeutic target. Among the tumor-forming cells, cancer cells, cancer-associated fibroblast (CAF), and tumor-infiltrating lymphocytes (TILs) comprise major proportion and players in tumor biology. Therefore, an in vitro tumor model composed of at least above three major cellular players better mimics a patient’s tumor. Here, we developed a patient-derived tumor model containing tumoroids, CAFs and TILs isolated from the patient’s tumor tissue by using one-stop isolation culture system. Our one-stop culture system utilizes a Transwell-based method that concurrently segregates cancer cells, CAFs, and TILs based on distinct characteristics such as cell size, migration speed, and growth. Lung tumoroids, fibroblasts, and TILs segregated in Matrigel-coated upper Transwell chamber, Transwell pores and in the lower chamber, respectively. Lung tumoroids maintain the patient’s lung cancer tissue characteristics and genetic features during extended in vitro cultivation. Fibroblasts extracted from the Transwell pores exhibit significant similarity to CAFs found in patient tissue. TILs in the lower part of the Transwell chamber closely resemble those found in the original cancer tissue analyzed by FACS. We have also refined the functionality of TILs through a two-week expansion process. Patients’ tumor composed of 0.15 to 95.3 % of EpCAM positive tumor cells, 0.14 to 19.4 % of CD90 positive fibroblasts and 3.98 to 91.2 % of immune cells in FACS analysis. By reassembling three main cellular components of the tumor in a proper proportion, we developed a co-culture platform for a patient-derived tumor model reproducing patient-specific TME conditions. The co-culture platform showed a potential not only for an experimental model but also to use a predictive test of personalized immunotherapy responses for lung cancer patients. Citation Format: YOO RI KO, Sung Min Kim, Hye Seon Park, Se Jin Jang. A comprehensive in vitro tumor model: One-stop simultaneous isolation and culture of tumoroids, CAFs and TILs for advancing immuno-oncology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4240.

Abstract 5334: Humanization of NOG mice and next-generation NOG strains to induce lineage-specific differentiation of immune cells for assessment of novel immune cell therapies, check point inhibitors, and immune cell engagers for translational immuno-oncology research

Abstract Background: The preclinical evaluation of novel immune therapies requires humanized immune system (HIS) mouse models. In previous studies we have demonstrated that either peripheral blood mononuclear cells (PBMC), subsets of PBMCs like T and NK cells or CD34+ hematopoietic stem cells (HSC) can be used to establish a HIS model. With the development of next-generation NOG mice, lineage-specific differentiation of immune cell sub-populations of interest can be supported. Transplantation of cell line-derived (CDX) or patient-derived (PDX) tumor xenografts on HIS mice provides a full model for human tumors for the investigation of checkpoint inhibitors (CPI), as well as novel cell therapies and immune cell engagers. Methods: HSC-humanized mice were generated by i.v. transplantation of CD34+ stem cells to immunodeficient mice. For humanization, NOG mice and next-generation NOG strains were used: NOG, NOG-EXL, hIL-2 NOG, hIL-6 NOG and FcResolv™ NOG mice were compared to each other for lineage-specific differentiation using single donors or a mixed HSC donor pool. Engraftment of immune cells was monitored by FACS analysis of blood every four weeks. In humanized NOG mice, CDX and PDX from different entities were s.c. transplanted and used to evaluate CPI. Results: Humanized hIL-2 NOG mice showed significantly decreased survival after HSC transplantation in comparison to the other mouse strains and had to be sacrificed within the first 6-8 weeks after HSC transplantation. In the other mouse strains, transplanted HSCs engrafted and differentiated mainly into B and T cells. NOG-EXL mice displayed the highest engraftment, with up to 80% of human cells in the blood, including a higher portion of myeloid cells. In humanized hIL-6 NOG mice a higher portion of monocytes could be determined. FcResolv™ NOG mice lack murine Fc gamma receptors in order to reduce confounding interactions between residual murine immune cells and antibody-based therapies, and they engraft and differentiate human HSCs similarly to the parent NOG strain. In humanized NOG mice, selected CDX and PDX tumors successfully engrafted without significant differences in tumor growth compared to non-humanized mice. CPI treatments induced tumor growth delay in selected models. Conclusions: Next-generation NOG mouse strains are characterized by a lineage-specific differentiation of immune cells depending on integrated human cytokines. Furthermore, we established a human tumor-immune-cell model for NOG mice using different entities of CDX or PDX in combination with CPI. Our human tumor-immune cell models allow preclinical translational studies on tumor immune biology as well as evaluation of new therapies, drug combinations and biomarker identification and validation. Citation Format: Maria Stecklum, Louise Baskin, Jens Hoffmann. Humanization of NOG mice and next-generation NOG strains to induce lineage-specific differentiation of immune cells for assessment of novel immune cell therapies, check point inhibitors, and immune cell engagers for translational immuno-oncology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5334.

Vulvar Carcinoma: Standard of Care and Perspectives.

Treatment of vulvar carcinoma (VC) is challenging. The objectives of this review were to describe for clinicians the epidemiologic and clinical aspects of VC, the standard of care in terms of primary local treatment and systemic therapies, and the recent innovations and perspectives emerging from translational research in immuno-oncology. We conducted a comprehensive review outlying the clinical aspects and biologic background of vulvar cancer, highlighting modern treatment strategies on the basis of a personalized approach. Epidemiologic data showed a recent rise in incidence of VC, attributed to human papillomavirus. Surgery is the mainstay of primary treatment, but multimodal approaches are frequently required in the presence of adverse prognosis histopathologic factors. Chemoradiation is indicated when organ-sparing surgery is not feasible. However, inability to achieve high locoregional control rates in advanced cases and the morbidity associated with local treatments are still key issues. Recent clinical data showed the benefit of individualized strategies combining organ-sparing surgical strategies, less invasive lymph node staging procedures, and refinement in radiotherapy modalities. Among the most important research area, there is a sound rationale for testing modern systemic approaches such as immune checkpoint inhibitors in selected patients with recurrent and/or metastatic tumors. Although no specific data exist for VC, the role of supportive care and post-treatment rehabilitation strategies is also crucial. There are still insufficient studies dedicated to patients with VC. Public health programs for prevention, screening, and early diagnosis are required, and clinical research should be strengthened to provide high-quality clinical evidence and improve patients' oncologic and functional outcomes.

Organoids: new frontiers in tumor immune microenvironment research.

The tumor microenvironment (TME) contains cells that regulate medication response and cancer growth in a major way. Tumor immunology research has been rejuvenated and cancer treatment has been changed by immunotherapy, a rapidly developing therapeutic approach. The growth patterns of tumor cells in vivo and the heterogeneity, complexity, and individuality of tumors produced from patients are not reflected in traditional two-dimensional tumor cell profiles. On the other hand, an in vitro three-dimensional (3D) model called the organoid model is gaining popularity. It can replicate the physiological and pathological properties of the original tissues in vivo. Tumor cells are the source of immune organoids. The TME characteristics can be preserved while preserving the variety of tumors by cultivating epithelial tumor cells with various stromal and immunological components. In addition to having genetic and physical similarities to human diseases and the ability to partially reconstruct the complex structure of tumors, these models are now widely used in research fields including cancer, developmental biology, regenerative mechanisms, drug development, disease modeling, and organ transplantation. This study reviews the function of organoids in immunotherapy and the tumor immune milieu. We also discuss current developments and suggest translational uses of tumor organoids in immuno-oncology research, immunotherapy modeling, and precision medicine.

A Microdissection Protocol for Proteogenomic Analysis of Histological Sections to Advance Drug Development.

Combining proteogenomics with laser capture microdissection (LCM) in cancer research offers a targeted way to explore the intricate interactions between tumor cells and the different microenvironment components. This is especially important for immuno-oncology (IO) research where improvements in the predictability of IO-based drugs are sorely needed, and depends on a better understanding of the spatial relationships involving the tumor, blood supply, and immune cell interactions, in the context of their associated microenvironments. LCM is used to isolate and obtain distinct histological cell types, which may be routinely performed on complex and heterogeneous solid tumor specimens. Once cells have been captured, nucleic acids and proteins may be extracted for in-depth multimodality molecular profiling assays. Optimizing the minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, RNA-seq may be performed for gene expression and DNA sequencing performed for the discovery and analysis of actionable mutations, copy number variation, methylation profiles, etc. However, there remains a need for highly sensitive proteomic methods targeting small-sized samples. A significant part of this protocol is an enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. Included is a detailed in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Described is a proteogenomic approach leveraged from microdissected fresh frozen tissue. The protocols may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.

Pathways to hypermutation in high-grade gliomas: Mechanisms, syndromes, and opportunities for immunotherapy.

Despite rapid advances in the field of immunotherapy, including the success of immune checkpoint inhibition in treating multiple cancer types, clinical response in high-grade gliomas (HGGs) has been disappointing. This has been in part attributed to the low tumor mutational burden (TMB) of the majority of HGGs. Hypermutation is a recently characterized glioma signature that occurs in a small subset of cases, which may open an avenue to immunotherapy. The substantially elevated TMB of these tumors most commonly results from alterations in the DNA mismatch repair pathway in the setting of extensive exposure to temozolomide or, less frequently, from inherited cancer predisposition syndromes. In this review, we discuss the genetics and etiology of hypermutation in HGGs, with an emphasis on the resulting genomic signatures, and the state and future directions of immuno-oncology research in these patient populations.

PDL1 immunohistochemistry in canine neoplasms: Validation of commercial antibodies, standardization of evaluation, and scoring systems.

Immuno-oncology research has brought to light the paradoxical role of immune cells in the induction and elimination of cancer. Programmed cell death protein 1 (PD1), expressed by tumor-infiltrating lymphocytes, and programmed cell death ligand 1 (PDL1), expressed by tumor cells, are immune checkpoint proteins that regulate the antitumor adaptive immune response. This study aimed to validate commercially available PDL1 antibodies in canine tissue and then, applying standardized methods and scoring systems used in human pathology, evaluate PDL1 immunopositivity in different types of canine tumors. To demonstrate cross-reactivity, a monoclonal antibody (22C3) and polyclonal antibody (cod. A1645) were tested by western blot. Cross-reactivity in canine tissue cell extracts was observed for both antibodies; however, the polyclonal antibody (cod. A1645) demonstrated higher signal specificity. Canine tumor histotypes were selected based on the human counterparts known to express PDL1. Immunohistochemistry was performed on 168 tumors with the polyclonal anti-PDL1 antibody. Only membranous labeling was considered positive. PDL1 labeling was detected both in neoplastic and infiltrating immune cells. The following tumors were immunopositive: melanomas (17 of 17; 100%), renal cell carcinomas (4 of 17; 24%), squamous cell carcinomas (3 of 17; 18%), lymphomas (2 of 14; 14%), urothelial carcinomas (2 of 18; 11%), pulmonary carcinomas (2 of 20; 10%), and mammary carcinomas (1 of 31; 3%). Gastric (0 of 10; 0%) and intestinal carcinomas (0 of 24; 0%) were negative. The findings of this study suggest that PDL1 is expressed in some canine tumors, with high prevalence in melanomas.

A bibliometric analysis of immuno-oncology research: Is a new global leader emerging?

e13593 Background: The last decade has witnessed an increase in cancer research globally, and a transformation of the anti-cancer armamentarium. The biggest innovation has been development of immuno-oncology (IO) therapies, and specifically immune checkpoint inhibitors (ICIs), targeting CTLA-4 and PD-(L)1, reshaping treatment paradigms for many cancers (e.g., melanoma). Since the first ICI approval in 2011, research from countries in the European Union (EU) and North America has dominated this field. However, more recently research authored in China has emerged as a potential leader. In this study we analysed cancer and IO research outputs since 2011, exploring emerging national trends, and focusing on ICI development. Methods: IO research articles were identified using a high-resolution bibliometric method, previously validated. Relevant articles were identified from the Web of Science, utilising a complex search strategy of pre-defined keywords, including the 9 FDA-approved ICI and 8 ICI approved in China-only. Additional search filters were used to determine secondary characteristics including country (assigned a fractional count based on proportional authorship), collaborations, and tumour site. Results: Since 2011, over 175,000 cancer research articles have been published globally. The number of articles published annually has increased steadily, largely driven by increasing outputs from Chinese authors. China is now the leading global publisher of cancer research accounting for 18% of all outputs in 2021, and has consistently surpassed US and EU total outputs since 2019. Over the past decade there has been a steady increase in total IO research outputs, rising by 378% (n = 2926) compared to 2011. China began publishing significant IO research in 2014 and has risen rapidly to dominate this field. In 2021, China accounted for 37% of total IO outputs. The US was previously dominant, however there has been a steady decline in proportional outputs from 63% in 2012-12 to 29% in 2019-21. Only 17% of published Chinese IO research is internationally collaborative. Further, Chinese research focuses on tumour sites with a high national prevalence, such as oesophagus, stomach and liver, dominating research outputs in these areas. Research on the 9 FDA-approved ICI is dominated by the US, with only 7% having Chinese author contribution. Conversely, the 8 ICIs approved in China-only have negligible (< 5%) author contributions from the US and EU. Conclusions: The past decade has witnessed a substantial rise in both cancer and IO research. IO articles published by Chinese authors, frequently exclusively, now account for over a third of annual outputs. Those authored in China focus on cancers with a high national prevalence, and link to ICIs approved exclusively in China. With increasing global appetite for ICIs, especially from middle-income countries, ICIs developed in China may offer a viable alternative to current options.

Seven-colour multiplex immunochemistry/immunofluorescence and whole slide imaging of frozen sections

Multiplex Immunochemistry/Immunofluorescence (mIHC/IF) aims to visualise multiple biomarkers in a single tissue section and is especially powerful when used on slide scanners coupled with digital analysis tools. mIHC/IF is commonly employed in immuno-oncology to characterise features of the tumour microenvironment (TME) and correlate them with clinical parameters to guide prognostication and therapy. However, mIHC/IF can be applied to a wide range of organisms in any physiological or disease context. Recent innovation has extended the number of markers that can be detected using slide scanners well beyond the 3–4 markers typically reported in traditional fluorescence microscopy. However, these methods often require sequential antibody staining and stripping, and are not compatible with frozen tissue sections.Using fluorophore-conjugated antibodies, we have established a simple mIHC/IF imaging workflow that enables simultaneous staining and detection of seven markers in a single section of frozen tissue. Coupled with automated whole slide imaging and digital quantification, our data efficiently revealed the tumour-immune complexity in metastatic melanoma. Computational image analysis quantified the immune and stromal cell populations present in the TME as well as their spatial interactions. This imaging workflow can also be performed with an indirect labelling panel consisting of primary and secondary antibodies. Our new methods, combined with digital quantification, will provide a valuable tool for high-quality mIHC/IF assays in immuno-oncology research and other translational studies, especially in circumstances where frozen sections are required for detection of particular markers, or for applications where frozen sections may be preferred, such as spatial transcriptomics.

Development of an Ultrahigh-plex Antibody Panel for Spatial Phenotyping of Murine Cancer Models

Abstract Single cell spatial phenotyping of protein biomarkers is an established tool for cancer researchers aiming to understand the biology of the tumor microenvironment. This technology has been developed around human tissues and there remains a paucity of applications available to study murine cancer models. Yet, mouse models are widely used for immuno-oncology (IO) research, justifying the development of spatial phenotyping applications for use in mice. We developed a >30-plex mouse IO antibody panel by selecting biomarkers for tumor, immune cell lineages, immune activation and checkpoints, as well as proliferation and structural markers. Purified antibodies, previously IHC validated in mouse models using rigorous protocols developed by Cell Signaling Technology (CST), were then conjugated to PhenoCycler barcodes, using a standardized conjugation protocol, and confirmed to show positive staining. We then performed ultrahigh-plex imaging using this IO antibody panel on the PhenoCycler-Fusion Platform, using murine lymphoid organs and/or tumor tissues. The resulting data were analyzed for cellular phenotypic compositions as well as the presence of cellular neighborhoods. The work presented here demonstrates the feasibility of conducting ultrahigh-plex spatial phenotyping in FFPE mouse tissues. The tools that were developed as part of this study can readily be deployed for discovery and translational research and will engender future comparative mouse and human spatial phenotyping studies that can further our understanding of cancer biology.

Design and Validation of Ultrahigh-plex Discovery Panels for Immune Checkpoint Targets

Abstract Ultrahigh-plex single-cell spatial phenotyping is revolutionizing immuno-oncology research. To enable in-depth characterization of the tumor microenvironment (TME), we have developed and validated pre-optimized antibody panels on cancer tissues to reveal key cell types and cellular architecture using the PhenoCycler™-Fusion system. We designed multiple panels that answer specific biological questions about the TME, and once multiplexed together create a comprehensive view of the structure and function of the TME. The resulting PhenoCode™ Discovery panels include markers that identify immune cell types, activation states, immune checkpoints, and mediators of proliferation. Each antibody in the panel was conjugated to an oligo barcode and stained on human FFPE tissue. Staining specificity of each conjugated antibody was assessed by comparing immunofluorescent results from PhenoCycler™ with a DAB chromogenic immunohistochemistry assay. The slide stained with a PhenoCode™ Discovery panel was imaged on a PhenoCycler™-Fusion platform, where dye-labeled oligo reporters (complementary to the barcodes) are hybridized to the barcode to visualize the antibody. Antibody concentration, exposure time, and corresponding dye were further optimized, verified, and validated as a whole module using tonsil and cancer tissues based on image analysis and intensity analysis. In this study, we showcased the rigorous process used for development and validation of PhenoCode™ Discovery panels and demonstrated how they can work together to collect comprehensive data from one tissue sample. The introduction of these pre-optimized antibody panels marks a pivotal point in the evolution of ultrahigh-plex spatial phenotyping.