Immunotherapy response induces divergent tertiary lymphoid structure morphologies in hepatocellular carcinoma.

Tertiary lymphoid structures (TLS) are associated with improved response in solid tumors treated with immune checkpoint blockade, but understanding of the prognostic and predictive value of TLS and the circumstances of their resolution is incomplete. Here we show that in hepatocellular carcinoma treated with neoadjuvant immunotherapy, high intratumoral TLS density at the time of surgery is associated with pathologic response and improved relapse-free survival. In areas of tumor regression, we identify a noncanonical involuted morphology of TLS marked by dispersion of the B cell follicle, persistence of a T cell zone enriched for T cell-mature dendritic cell interactions and increased expression of T cell memory markers. Collectively, these data suggest that TLS can serve as both a prognostic and predictive marker of response to immunotherapy in hepatocellular carcinoma and that late-stage TLS may support T cell memory formation after elimination of a viable tumor.

Spatial multi-omics reveal intratumoral humoral immunity niches associated with tertiary lymphoid structures in pancreatic cancer immunotherapy pathologic responders.

Pancreatic adenocarcinoma (PDAC) is a rapidly progressing cancer that responds poorly to immunotherapies. Intratumoral tertiary lymphoid structures (TLS) have been associated with rare long-term PDAC survivors, but the role of TLS in PDAC and their spatial relationships within the context of the broader tumor microenvironment remain unknown. We generated a spatial multi-omics atlas encompassing 26 PDAC tumors from patients treated with combination immunotherapies. Using machine learning-enabled H&E image classification models and unsupervised gene expression matrix factorization methods for spatial transcriptomics, we characterized cellular states within TLS niches spanning across distinct morphologies and immunotherapies. Unsupervised learning generated a TLS-specific spatial gene expression signature that significantly associates with improved survival in PDAC patients. These analyses demonstrate TLS-associated intratumoral B cell maturation in pathological responders, confirmed with spatial proteomics and BCR profiling. Our study also identifies spatial features of pathologic immune responses, revealing TLS maturation colocalizing with IgG/IgA distribution and extracellular matrix remodeling. HIGHLIGHTS: Integrated multi-modal spatial profiling of human PDAC tumors from neoadjuvant immunotherapy clinical trials reveal diverse spatial niches enriched in TLS.TLS maturity is influenced by tumor location and the cellular neighborhoods in which TLS immune cells are recruited.Unsupervised machine learning of genome-wide signatures on spatial transcriptomics data characterizes the TLS-enriched TME and associates TLS transcriptomes with survival outcomes in PDAC.Interactions of spatially variable gene expression patterns showed TLS maturation is coupled with immunoglobulin distribution and ECM remodeling in pathologic responders.Intratumoral plasma cell and immunoglobin gene expression spatial dynamics demonstrate trafficking of TLS-driven humoral immunity in the PDAC TME. Significance: We report a spatial multi-omics atlas of PDAC tumors from a series of immunotherapy neoadjuvant clinical trials. Intratumorally, pathologic responders exhibit mature TLS that propagate plasma cells into malignant niches. Our findings offer insights on the role of TLS-associated humoral immunity and stromal remodeling during immunotherapy treatment.

Immune-related events in individuals with solid tumors on immunotherapy associate with Th17 and Th2 signatures.

BACKGROUNDImmune-related adverse events (irAEs) and their associated morbidity/mortality are a key concern for patients receiving immune checkpoint inhibitors (ICIs). Prospective evaluation of the drivers of irAEs in a diverse pan-tumor cohort is needed to identify patients at greatest risk and to develop rational treatment and interception strategies.METHODSIn an observational study, we prospectively collected blood samples and performed regular clinical evaluations for irAEs in patients receiving ICI therapy as standard of care for solid tumors. We performed in-parallel analysis of cytokines by Luminex immunoassay and circulating immune cells by cytometry by time-of-flight (CyTOF) at baseline and on treatment to investigate mechanisms of irAEs.RESULTSWe enrolled 111 patients, of whom 40.5% developed a symptomatic irAE (grade ≥ 2). Development of a grade ≥ 2 irAE was positively associated with the use of combination ICI and a history of an autoimmune disorder. Early changes in T helper 17 (Th17) (IL-6, IL-17f), type 2 (IL-5, IL-13, IL-25), and type 1 (TNF-α) cytokine signatures and congruent on-treatment expansions of Th17 and Th2 effector memory (Th2EM) T cell populations in peripheral blood were positively associated with the development of grade ≥2 irAEs. IL-6 levels were also associated with inferior cancer-specific survival and overall survival.CONCLUSIONSIn a diverse, prospective pan-tumor cohort, Th17 and Th2 skewing during early ICI treatment was associated with the development of clinically relevant irAEs but not antitumor responses, providing possible targets for monitoring and therapeutic interventions.FUNDINGJohns Hopkins Bloomberg-Kimmel Institute for Cancer Immunotherapy, the NCI SPORE in Gastrointestinal Cancers (P50 CA062924), NCI grant (R50CA243627 to LD), the NIH Center Core Grant (P30 CA006973), Swim Across America (to MY), NIAMS (K23AR075872 to LC), and imCORE-Genentech grant 137515 (to Johns Hopkins Medicine on behalf of MY).

PanIN and CAF transitions in pancreatic carcinogenesis revealed with spatial data integration.

This study introduces a new imaging, spatial transcriptomics (ST), and single-cell RNA-sequencing integration pipeline to characterize neoplastic cell state transitions during tumorigenesis. We applied a semi-supervised analysis pipeline to examine premalignant pancreatic intraepithelial neoplasias (PanINs) that can develop into pancreatic ductal adenocarcinoma (PDAC). Their strict diagnosis on formalin-fixed and paraffin-embedded (FFPE) samples limited the single-cell characterization of human PanINs within their microenvironment. We leverage whole transcriptome FFPE ST to enable the study of a rare cohort of matched low-grade (LG) and high-grade (HG) PanIN lesions to track progression and map cellular phenotypes relative to single-cell PDAC datasets. We demonstrate that cancer-associated fibroblasts (CAFs), including antigen-presenting CAFs, are located close to PanINs. We further observed a transition from CAF-related inflammatory signaling to cellular proliferation during PanIN progression. We validate these findings with single-cell high-dimensional imaging proteomics and transcriptomics technologies. Altogether, our semi-supervised learning framework for spatial multi-omics has broad applicability across cancer types to decipher the spatiotemporal dynamics of carcinogenesis.

Impact of Neoadjuvant Immunotherapy on Recurrence-Free Survival in Patients with High-Risk Localized HCC.

Surgical resection for localized hepatocellular carcinoma (HCC) is typically reserved for a minority of patients with favorable tumor features and anatomy. Neoadjuvant immunotherapy can expand the number of patients who are candidates for surgical resection and potentially reduce the chance for recurrence, but its role in HCC not defined. We retrospectively examined the outcomes of patients who underwent surgical resection for HCC at the Johns Hopkins Hospital and compared the clinical outcomes of patients who received neoadjuvant immunotherapy with those who underwent upfront resection. The clinical cohort included a total of 92 patients, 36 of whom received neoadjuvant immune checkpoint inhibitor (ICI)-based treatment. A majority of patients (61.1%) who received neoadjuvant ICI-based therapy were outside of standard resectability criteria and were more likely to have features known to confer risk of disease recurrence, including α-fetoprotein ≥ 400 ng/mL (P = 0.02), tumor diameter ≥ 5 cm (P = 0.001), portal vein invasion (P < 0.001), and multifocality (P < 0.001). Patients who received neoadjuvant immunotherapy had similar rates of margin-negative resection (P = 0.47) and recurrence-free survival (RFS) as those who underwent upfront surgical resection (median RFS 44.8 months compared with 49.3 months, respectively, log-rank P = 0.66). There was a nonsignificant trend toward superior RFS in the subset of patients with a pathologic response (tumor necrosis ≥ 70%) with neoadjuvant immunotherapy. Neoadjuvant ICI-based therapy may allow high-risk patients, including those who are outside traditional resectability criteria, to achieve comparable clinical outcomes with those who undergo upfront resection. SIGNIFICANCE: Surgical resection for localized HCC is typically only reserved for those with solitary tumors without vascular invasion. In this retrospective analysis, we show that neoadjuvant immunotherapy may allow high-risk patients, including those who are outside of standard resection criteria, to undergo successful margin-negative resection and achieve comparable long-term clinical outcomes compared with upfront resection. These findings highlight need for prospective studies on neoadjuvant immunotherapy in HCC.

Phosphodiesterase-5 inhibition collaborates with vaccine-based immunotherapy to reprogram myeloid cells in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is highly lethal and resistant to immunotherapy. Although immune recognition can be enhanced with immunomodulatory agents including checkpoint inhibitors and vaccines, few patients experience clinical efficacy because the tumor immune microenvironment (TiME) is dominated by immunosuppressive myeloid cells that impose T cell inhibition. Inhibition of phosphodiesterase-5 (PDE5) was reported to downregulate metabolic regulators arginase and inducible NOS in immunosuppressive myeloid cells and enhance immunity against immune-sensitive tumors, including head and neck cancers. We show for the first time to our knowledge that combining a PDE5 inhibitor, tadalafil, with a mesothelin-specific vaccine, anti-programmed cell death protein 1, and anti-cytotoxic T lymphocyte-associated protein 4 yields antitumor efficacy even against immune-resistant PDAC. To determine immunologic advantages conferred by tadalafil, we profiled the TiME using mass cytometry and single-cell RNA-sequencing analysis with Domino to infer intercellular signaling. Our analyses demonstrated that tadalafil reprograms myeloid cells to be less immunosuppressive. Moreover, tadalafil synergized with the vaccine, enhancing T cell activation including mesothelin-specific T cells. Tadalafil treatment was also associated with myeloid/T cell signaling axes important for antitumor responses (e.g., Cxcr3, Il12). Our study shows that PDE5 inhibition combined with vaccine-based immunotherapy promotes pro-inflammatory states of myeloid cells, activation of T cells, and enhanced myeloid/T cell crosstalk to yield antitumor efficacy against immune-resistant PDAC.

Mapping the breast tumor microenvironment: proximity analysis reveals spatial relationships between macrophage subtypes and metastasis-initiating cancer cells.

Metastasis is responsible for the majority of cancer-related fatalities. We previously identified specific cancer cell populations responsible for metastatic events which are cytokeratin-14 (CK14) and E-cadherin positive in luminal tumors, and E-cadherin and vimentin positive in triple-negative tumors. Since cancer cells evolve within a complex ecosystem comprised of immune cells and stromal cells, we sought to decipher the spatial interactions of these aggressive cancer cell populations within the tumor microenvironment (TME). We used imaging mass cytometry to detect 36 proteins in tumor microarrays containing paired primary and metastatic lesions from luminal or triple-negative breast cancers (TNBC), resulting in a dataset of 1,477,337 annotated cells. Focusing on metastasis-initiating cell populations, we observed close proximity to specific fibroblast and macrophage subtypes, a relationship maintained between primary and metastatic tumors. Notably, high CK14 in luminal cancer cells and high vimentin in TNBC cells correlated with close proximity to specific macrophage subtypes (CD163intCD206intPDL1intHLA-DR+ or PDL1highARG1high). Our in-depth spatial analysis demonstrates that metastasis-initiating cancer cells consistently colocalizes with distinct cell populations within the TME, suggesting a role for these cell-cell interactions in promoting metastasis.

Computational methods and biomarker discovery strategies for spatial proteomics: a review in immuno-oncology.

Advancements in imaging technologies have revolutionized our ability to deeply profile pathological tissue architectures, generating large volumes of imaging data with unparalleled spatial resolution. This type of data collection, namely, spatial proteomics, offers invaluable insights into various human diseases. Simultaneously, computational algorithms have evolved to manage the increasing dimensionality of spatial proteomics inherent in this progress. Numerous imaging-based computational frameworks, such as computational pathology, have been proposed for research and clinical applications. However, the development of these fields demands diverse domain expertise, creating barriers to their integration and further application. This review seeks to bridge this divide by presenting a comprehensive guideline. We consolidate prevailing computational methods and outline a roadmap from image processing to data-driven, statistics-informed biomarker discovery. Additionally, we explore future perspectives as the field moves toward interfacing with other quantitative domains, holding significant promise for precision care in immuno-oncology.

3D histology reveals that immune response to pancreatic precancers is heterogeneous and depends on global pancreas structure.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer for which few effective therapies exist. Immunotherapies specifically are ineffective in pancreatic cancer, in part due to its unique stromal and immune microenvironment. Pancreatic intraepithelial neoplasia, or PanIN, is the main precursor lesion to PDAC. Recently it was discovered that PanINs are remarkably abundant in the grossly normal pancreas, suggesting that the vast majority will never progress to cancer. Here, through construction of 48 samples of cm3-sized human pancreas tissue, we profiled the immune microenvironment of 1,476 PanINs in 3D and at single-cell resolution to better understand the early evolution of the pancreatic tumor microenvironment and to determine how inflammation may play a role in cancer progression. We found that bulk pancreatic inflammation strongly correlates to PanIN cell fraction. We found that the immune response around PanINs is highly heterogeneous, with distinct immune hotspots and cold spots that appear and disappear in a span of tens of microns. Immune hotspots generally mark locations of higher grade of dysplasia or locations near acinar atrophy. The immune composition at these hotspots is dominated by naïve, cytotoxic, and regulatory T cells, cancer associated fibroblasts, and tumor associated macrophages, with little similarity to the immune composition around less-inflamed PanINs. By mapping FOXP3+ cells in 3D, we found that regulatory T cells are present at higher density in larger PanIN lesions compared to smaller PanINs, suggesting that the early initiation of PanINs may not exhibit an immunosuppressive response. This analysis demonstrates that while PanINs are common in the pancreases of most individuals, inflammation may play a pivotal role, both at the bulk and the microscopic scale, in demarcating regions of significance in cancer progression.

High-motility pro-tumorigenic monocytes drive macrophage enrichment in the tumor microenvironment.

Enrichment of tumor-associated macrophages (TAMΦs) in the tumor microenvironment correlates with worse clinical outcomes in triple-negative breast cancer (TNBC) patients, prompting the development of therapies to inhibit TAMΦ infiltration. However, the lackluster efficacy of CCL2-based chemotaxis blockade in clinical trials suggests that a new understanding of monocyte/macrophage infiltration may be necessary. Here we demonstrate that random migration, and not only chemotaxis, drives macrophage tumor infiltration. We identified tumor- associated monocytes (TAMos) that display a dramatically enhanced migration capability, induced rapidly by the tumor microenvironment, that drives effective tumor infiltration, in contrast to low-motility differentiated macrophages. TAMo, not TAMΦ, promotes cancer cell proliferation through activation of the MAPK pathway. IL-6 secreted both by cancer cells and TAMo themselves enhances TAMo migration by increasing dendritic protrusion dynamics and myosin- based contractility via the JAK2/STAT3 signaling pathway. Independent from CCL2 mediated chemotaxis, IL-6 driven enhanced migration and pro-proliferative effect of TAMo were validated in a syngeneic TNBC mouse model. Depletion of IL-6 in cancer cells significantly attenuated monocyte infiltration and reversed TAMo-induced cancer cell proliferation. This work reveals the critical role random migration plays in monocyte driven TAMΦ enrichment in a tumor and pinpoints IL-6 as a potential therapeutic target in combination with CCL2 to ameliorate current strategies against TAMΦ infiltration.

Natural killer cells associate with epithelial cells in the pancreatic ductal adenocarcinoma tumor microenvironment.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer. PDAC's poor prognosis and resistance to immunotherapy are attributed in part to its dense, fibrotic tumor microenvironment (TME), which is known to inhibit immune cell infiltration. We recently demonstrated that PDAC patients with higher natural killer (NK) cell content and activation have better survival rates. However, NK cell interactions in the PDAC TME have yet to be deeply studied. We show here that NK cells are present and active in the human PDAC TME. Methods: We used imaging mass cytometry (IMC) to assess NK cell content, function, and spatial localization in human PDAC samples. Then, we used CellChat, a tool to infer ligand-receptor interactions, on a human PDAC scRNAseq dataset to further define NK cell interactions in PDAC. Results: Spatial analyses showed for the first time that active NK cells are present in the PDAC TME, and both associate and interact with malignant epithelial cell ducts. We also found that fibroblast-rich, desmoplastic regions limit NK cell infiltration in the PDAC TME. CellChat analysis identified that the CD44 receptor on NK cells interacts with PDAC extracellular matrix (ECM) components such as collagen, fibronectin and laminin expressed by fibroblasts and malignant epithelial cells. This led us to hypothesize that these interactions play roles in regulating NK cell motility in desmoplastic PDAC TMEs. Using 2D and 3D in vitro assays, we found that CD44 neutralization significantly increased NK cell invasion through matrix. Conclusions: Targeting ECM-immune cell interactions may increase NK cell invasion into the PDAC TME.

Integration of Clinical Trial Spatial Multiomics Analysis and Virtual Clinical Trials Enables Immunotherapy Response Prediction and Biomarker Discovery.

Due to the lack of treatment options, there remains a need to advance new therapeutics in hepatocellular carcinoma (HCC). The traditional approach moves from initial molecular discovery through animal models to human trials to advance novel systemic therapies that improve treatment outcomes for patients with cancer. Computational methods that simulate tumors mathematically to describe cellular and molecular interactions are emerging as promising tools to simulate the impact of therapy entirely in silico, potentially greatly accelerating delivery of new therapeutics to patients. To facilitate the design of dosing regimens and identification of potential biomarkers for immunotherapy, we developed a new computational model to track tumor progression at the organ scale while capturing the spatial heterogeneity of the tumor in HCC. This computational model of spatial quantitative systems pharmacology was designed to simulate the effects of combination immunotherapy. The model was initiated using literature-derived parameter values and fitted to the specifics of HCC. Model validation was done through comparison with spatial multiomics data from a neoadjuvant HCC clinical trial combining anti-PD1 immunotherapy and a multitargeted tyrosine kinase inhibitor cabozantinib. Validation using spatial proteomics data from imaging mass cytometry demonstrated that closer proximity between CD8 T cells and macrophages correlated with nonresponse. We also compared the model output with Visium spatial transcriptomics profiling of samples from posttreatment tumor resections in the clinical trial and from another independent study of anti-PD1 monotherapy. Spatial transcriptomics data confirmed simulation results, suggesting the importance of spatial patterns of tumor vasculature and TGFß in tumor and immune cell interactions. Our findings demonstrate that incorporating mathematical modeling and computer simulations with high-throughput spatial multiomics data provides a novel approach for patient outcome prediction and biomarker discovery. Significance: Incorporating mathematical modeling and computer simulations with high-throughput spatial multiomics data provides an effective approach for patient outcome prediction and biomarker discovery.

Spatial Mass Cytometry-Based Single-Cell Imaging Reveals a Disrupted Epithelial-Immune Axis in Prurigo Nodularis.

Prurigo nodularis (PN) is a chronic, inflammatory skin condition that disproportionately affects African Americans and features intensely pruritic, hyperkeratotic nodules on the extremities and trunk. PN is understudied compared with other inflammatory skin diseases, with the spatial organization of the cutaneous infiltrate in PN yet to be characterized. In this work, we employ spatial imaging mass cytometry to visualize PN lesional skin inflammation and architecture with single-cell resolution through an unbiased machine learning approach. PN lesional skin has increased expression of caspase 3, NF-kB, and phosphorylated signal transducer and activator of transcription 3 compared with healthy skin. Keratinocytes in lesional skin are subdivided into CD14+CD33+, CD11c+, CD63+, and caspase 3-positive innate subpopulations. CD14+ macrophage populations expressing phosphorylated extracellular signal-regulated kinase 1/2 correlate positively with patient-reported itch (P = .006). Hierarchical clustering reveals a cluster of patients with PN with greater atopy, increased NF-kB+ signal transducer and activator of transcription 3-positive phosphorylated extracellular signal-regulated kinase 1/2-positive monocyte-derived myeloid dendritic cells, and increased vimentin expression (P < .05). Neighborhood analysis finds interactions between CD14+ macrophages, CD3+ T cells, monocyte-derived myeloid dendritic cells, and keratinocytes expressing innate immune markers. These findings highlight phosphorylated extracellular signal-regulated kinase-positive CD14+ macrophages as contributors to itch and suggest an epithelial-immune axis in PN pathogenesis.

Multimodal immune phenotyping reveals microbial-T cell interactions that shape pancreatic cancer.

Microbes are an integral component of the tumor microenvironment. However, determinants of microbial presence remain ill-defined. Here, using spatial-profiling technologies, we show that bacterial and immune cell heterogeneity are spatially coupled. Mouse models of pancreatic cancer recapitulate the immune-microbial spatial coupling seen in humans. Distinct intra-tumoral niches are defined by T cells, with T cell-enriched and T cell-poor regions displaying unique bacterial communities that are associated with immunologically active and quiescent phenotypes, respectively, but are independent of the gut microbiome. Depletion of intra-tumoral bacteria slows tumor growth in T cell-poor tumors and alters the phenotype and presence of myeloid and B cells in T cell-enriched tumors but does not affect T cell infiltration. In contrast, T cell depletion disrupts the immunological state of tumors and reduces intra-tumoral bacteria. Our results establish a coupling between microbes and T cells in cancer wherein spatially defined immune-microbial communities differentially influence tumor biology.

Reprogramming the Intrahepatic Cholangiocarcinoma Immune Microenvironment by Chemotherapy and CTLA-4 Blockade Enhances Anti-PD-1 Therapy.

Intrahepatic cholangiocarcinoma (ICC) has limited therapeutic options and a dismal prognosis. Adding blockade of the anti-programmed cell death protein (PD)-1 pathway to gemcitabine/cisplatin chemotherapy has recently shown efficacy in biliary tract cancers but with low response rates. Here, we studied the effects of anti-cytotoxic T lymphocyte antigen (CTLA)-4 when combined with anti-PD-1 and gemcitabine/cisplatin in orthotopic murine models of ICC. This combination therapy led to substantial survival benefits and reduction of morbidity in two aggressive ICC models that were resistant to immunotherapy alone. Gemcitabine/cisplatin treatment increased tumor-infiltrating lymphocytes and normalized the ICC vessels and, when combined with dual CTLA-4/PD-1 blockade, increased the number of activated CD8+Cxcr3+IFNγ+ T cells. CD8+ T cells were necessary for the therapeutic benefit because the efficacy was compromised when CD8+ T cells were depleted. Expression of Cxcr3 on CD8+ T cells is necessary and sufficient because CD8+ T cells from Cxcr3+/+ but not Cxcr3-/- mice rescued efficacy in T cell‒deficient mice. Finally, rational scheduling of anti-CTLA-4 "priming" with chemotherapy followed by anti-PD-1 therapy achieved equivalent efficacy with reduced overall drug exposure. These data suggest that this combination approach should be clinically tested to overcome resistance to current therapies in ICC patients.

Single-Cell RNA Sequencing Reveals Dysregulated POSTN+WNT5A+ Fibroblast Subclusters in Prurigo Nodularis.

Prurigo nodularis (PN) is an intensely pruritic, inflammatory skin disease with a poorly understood pathogenesis. We performed single-cell transcriptomic profiling of 28,695 lesional and nonlesional PN cells. Lesional PN has increased dysregulated fibroblasts (FBs) and myofibroblasts. FBs in lesional PN were shifted toward a cancer-associated FB-like phenotype, with POSTN+WNT5A+ cancer-associated FBs increased in PN and similarly so in squamous cell carcinoma. A multicenter cohort study revealed an increased risk of squamous cell carcinoma and cancer-associated FB-associated malignancies (breast and colorectal) in patients with PN. Systemic fibroproliferative diseases (renal sclerosis and idiopathic pulmonary fibrosis) were upregulated in patients with PN. Ligand-receptor analyses demonstrated an FB neuronal axis with FB-derived WNT5A and periostin interactions with neuronal receptors melanoma cell adhesion molecule and ITGAV. These findings identify a pathogenic and targetable POSTN+WNT5A+ FB subpopulation that may predispose cancer-associated FB-associated malignancies in patients with PN.

Systems immunology spanning tumors, lymph nodes, and periphery.

The immune system defines a complex network of tissues and cell types that orchestrate responses across the body in a dynamic manner. The local and systemic interactions between immune and cancer cells contribute to disease progression. Lymphocytes are activated in lymph nodes, traffic through the periphery, and impact cancer progression through their interactions with tumor cells. As a result, therapeutic response and resistance are mediated across tissues, and a comprehensive understanding of lymphocyte dynamics requires a systems-level approach. In this review, we highlight experimental and computational methods that can leverage the study of leukocyte trafficking through an immunomics lens and reveal how adaptive immunity shapes cancer.

Preventing NK cell activation in the damaged liver induced by cabozantinib/PD-1 blockade increases survival in hepatocellular carcinoma models.

The addition of anti-VEGF antibody treatment to immune checkpoint blockade (ICB) has increased the efficacy of immunotherapy in advanced hepatocellular carcinoma (HCC). Despite an initial promise, adding multitargeted kinase inhibitors of VEGFR with ICB has failed to increase survival in HCC. To reveal the mechanisms underlying treatment failure, we studied the effects of cabozantinib/ICB using orthotopic murine HCC models with or without liver damage. We monitored tumor growth and liver function, recorded survival outcomes, and performed immune profiling studies for intra-tumoral and surrounding liver. Cabozantinib/ICB treatment led to tumor regression and significantly improved survival in mice with normal livers. However, consistent with the clinical findings, combination therapy failed to show survival benefits despite similar tumor control when tested in the same models but in mice with liver fibrosis. Moreover, preclinical and clinical data converged, showing that activating immune responses by cabozantinib/ICB treatment induced hepatoxicity. Immune profiling revealed that combination therapy effectively reprogrammed the tumor immune microenvironment and increased NK cell infiltration and activation in the damaged liver tissue. Surprisingly, systemic depletion of NK reduced hepatotoxicity elicited by the combination therapy without compromising its anti-cancer effect, and significantly enhanced the survival benefit even in mice with HCC and underlying liver fibrosis. These findings demonstrate that preventing NK activation allowed for maintaining a favorable therapeutic ratio when combining ICB with cabozantinib in advanced HCC models.

Immune landscape of tertiary lymphoid structures in hepatocellular carcinoma (HCC) treated with neoadjuvant immune checkpoint blockade.

Neoadjuvant immunotherapy is thought to produce long-term remissions through induction of antitumor immune responses before removal of the primary tumor. Tertiary lymphoid structures (TLS), germinal center-like structures that can arise within tumors, may contribute to the establishment of immunological memory in this setting, but understanding of their role remains limited. Here, we investigated the contribution of TLS to antitumor immunity in hepatocellular carcinoma (HCC) treated with neoadjuvant immunotherapy. We found that neoadjuvant immunotherapy induced the formation of TLS, which were associated with superior pathologic response, improved relapse free survival, and expansion of the intratumoral T and B cell repertoire. While TLS in viable tumor displayed a highly active mature morphology, in areas of tumor regression we identified an involuted TLS morphology, which was characterized by dispersion of the B cell follicle and persistence of a T cell zone enriched for ongoing antigen presentation and T cell-mature dendritic cell interactions. Involuted TLS showed increased expression of T cell memory markers and expansion of CD8+ cytotoxic and tissue resident memory clonotypes. Collectively, these data reveal the circumstances of TLS dissolution and suggest a functional role for late-stage TLS as sites of T cell memory formation after elimination of viable tumor.