Spatial oncology: Translating contextual biology to the clinic.

Microscopic examination of cells in their tissue context has been the driving force behind diagnostic histopathology over the past two centuries. Recently, the rise of advanced molecular biomarkers identified through single cell profiling has increased our understanding of cellular heterogeneity in cancer but have yet to significantly impact clinical care. Spatial technologies integrating molecular profiling with microenvironmental features are poised to bridge this translational gap by providing critical in situ context for understanding cellular interactions and organization. Here, we review how spatial tools have been used to study tumor ecosystems and their clinical applications. We detail findings in cell-cell interactions, microenvironment composition, and tissue remodeling for immune evasion and therapeutic resistance. Additionally, we highlight the emerging role of multi-omic spatial profiling for characterizing clinically relevant features including perineural invasion, tertiary lymphoid structures, and the tumor-stroma interface. Finally, we explore strategies for clinical integration and their augmentation of therapeutic and diagnostic approaches.

Integrating priorities at the intersection of cancer and neuroscience.

Cancer neuroscience is a rapidly growing multidisciplinary field that conceptualizes tumors as tissues fully integrated into the nervous system. Recognizing the complexity and challenges in this field is of fundamental importance to achieving the goal of translational impact for cancer patients. Our commentary highlights key scientific priorities, optimal training settings, and roadblocks to translating scientific findings to the clinic in this emerging field, aiming to formulate a transformative and cohesive path forward.

Spatially resolved analysis of pancreatic cancer identifies therapy-associated remodeling of the tumor microenvironment.

In combination with cell-intrinsic properties, interactions in the tumor microenvironment modulate therapeutic response. We leveraged single-cell spatial transcriptomics to dissect the remodeling of multicellular neighborhoods and cell-cell interactions in human pancreatic cancer associated with neoadjuvant chemotherapy and radiotherapy. We developed spatially constrained optimal transport interaction analysis (SCOTIA), an optimal transport model with a cost function that includes both spatial distance and ligand-receptor gene expression. Our results uncovered a marked change in ligand-receptor interactions between cancer-associated fibroblasts and malignant cells in response to treatment, which was supported by orthogonal datasets, including an ex vivo tumoroid coculture system. We identified enrichment in interleukin-6 family signaling that functionally confers resistance to chemotherapy. Overall, this study demonstrates that characterization of the tumor microenvironment using single-cell spatial transcriptomics allows for the identification of molecular interactions that may play a role in the emergence of therapeutic resistance and offers a spatially based analysis framework that can be broadly applied to other contexts.

Targeting heterogeneous tumor microenvironments in pancreatic cancer mouse models of metastasis by TGFß depletion.

The dual tumor-suppressive and promoting function of TGFß signaling has made its targeting challenging. We hereby examined the effects of TGFß depletion by AVID200/BMS-986416(TGFß-TRAP), a TGFß ligand trap, on the tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) murine models with different organ-specific metastasis. Our study demonstrated that TGFß-TRAP potentiates the efficacy of anti-PD-1 in a PDAC orthotopic murine model with liver metastasis tropism, significantly reducing liver metastases. We further demonstrated the heterogeneous response of cytotoxic effector T-cells to combination TGFß-TRAP and anti-PD-1 treatment across several tumor models. Single-nuclear RNA-sequencing suggested that TGFß-TRAP modulates cancer associated fibroblast (CAF) heterogeneity and suppresses neutrophil degranulation and CD4+ T-cell response to neutrophil degranulation. Ligand-receptor analysis indicated that TGFß-TRAP may modulate the CCL5-CCR5 axis as well as co-stimulatory and checkpoint signaling from CAFs and myeloid cells. Notably, the most highly expressed ligands of CCR5 shifted from the immunosuppressive CCL5 to CCL7 and CCL8, which may mediate the immune agonist activity of CCR5 following TGFß-TRAP and anti-PD-1 combination treatment. This study suggested that TGFß depletion modulates CAF heterogeneity and potentially reprograms CAFs and myeloid cells into anti-tumor immune agonists in PDAC, supporting the validation of such effects in human specimen.

Hypofractionated Radiotherapy-Related Lymphopenia Is Associated With Worse Survival in Unresectable Intrahepatic Cholangiocarcinoma.

OBJECTIVE: The aim of this study was to evaluate the incidence of radiotherapy (RT)-related lymphopenia, its predictors, and association with survival in unresectable intrahepatic cholangiocarcinoma (ICC) treated with hypofractionated-RT (HF-RT). METHODS: Retrospective analysis of 96 patients with unresectable ICC who underwent HF-RT (median 58.05 Gy in 15 fractions) between 2009 and 2022 was performed. Absolute lymphocyte count (ALC) nadir within 12 weeks of RT was analyzed. Primary variable of interest was severe lymphopenia, defined as Grade 3+ (ALC <0.5 k/µL) per CTCAE v5.0. Primary outcome of interest was overall survival (OS) from RT. RESULTS: Median follow-up was 16 months. Fifty-two percent of patients had chemotherapy pre-RT, 23% during RT, and 40% post-RT. Pre-RT, median ALC was 1.1 k/µL and 5% had severe lymphopenia. Post-RT, 68% developed RT-related severe lymphopenia. Patients who developed severe lymphopenia had a significantly lower pre-RT ALC (median 1.1 vs. 1.5 k/µL, P =0.01) and larger target tumor volume (median 125 vs. 62 cm 3 , P =0.02). In our multivariable Cox model, severe lymphopenia was associated with a 1.7-fold increased risk of death ( P =0.04); 1-year OS rates were 63% vs 77% ( P =0.03). Receipt of photon versus proton-based RT (OR=3.50, P =0.02), higher mean liver dose (OR=1.19, P <0.01), and longer RT duration (OR=1.49, P =0.02) predicted severe lymphopenia. CONCLUSIONS: HF-RT-related lymphopenia is an independent prognostic factor for survival in patients with unresectable ICC. Patients with lower baseline ALC and larger tumor volume may be at increased risk, and use of proton therapy, minimizing mean liver dose, and avoiding treatment breaks may reduce RT-related lymphopenia.

Sustained and Localized Drug Depot Release Using Radiation-Activated Scintillating Nanoparticles.

Clinical treatment of cancer commonly incorporates X-ray radiation therapy (XRT), and developing spatially precise radiation-activatable drug delivery strategies may improve XRT efficacy while limiting off-target toxicities associated with systemically administered drugs. Nevertheless, achieving this has been challenging thus far because strategies typically rely on radical species with short lifespans, and the inherent nature of hypoxic and acidic tumor microenvironments may encourage spatially heterogeneous effects. It is hypothesized that the challenge could be bypassed by using scintillating nanoparticles that emit light upon X-ray absorption, locally forming therapeutic drug depots in tumor tissues. Thus a nanoparticle platform (Scintillating nanoparticle Drug Depot; SciDD) that enables the local release of cytotoxic payloads only after activation by XRT is developed, thereby limiting off-target toxicity. As a proof-of-principle, SciDD is used to deliver a microtubule-destabilizing payload MMAE (monomethyl auristatin E). With as little as a 2 Gy local irradiation to tumors, MMAE payloads are released effectively to kill tumor cells. XRT-mediated drug release is demonstrated in multiple mouse cancer models and showed efficacy over XRT alone (p < 0.0001). This work shows that SciDD can act as a local drug depot with spatiotemporally controlled release of cancer therapeutics.

Therapy-associated remodeling of pancreatic cancer revealed by single-cell spatial transcriptomics and optimal transport analysis.

In combination with cell intrinsic properties, interactions in the tumor microenvironment modulate therapeutic response. We leveraged high-plex single-cell spatial transcriptomics to dissect the remodeling of multicellular neighborhoods and cell-cell interactions in human pancreatic cancer associated with specific malignant subtypes and neoadjuvant chemotherapy/radiotherapy. We developed Spatially Constrained Optimal Transport Interaction Analysis (SCOTIA), an optimal transport model with a cost function that includes both spatial distance and ligand-receptor gene expression. Our results uncovered a marked change in ligand-receptor interactions between cancer-associated fibroblasts and malignant cells in response to treatment, which was supported by orthogonal datasets, including an ex vivo tumoroid co-culture system. Overall, this study demonstrates that characterization of the tumor microenvironment using high-plex single-cell spatial transcriptomics allows for identification of molecular interactions that may play a role in the emergence of chemoresistance and establishes a translational spatial biology paradigm that can be broadly applied to other malignancies, diseases, and treatments.

It is better to light a candle than to curse the darkness: single-cell transcriptomics sheds new light on pancreas biology and disease.

Recent advances in single-cell RNA sequencing and bioinformatics have drastically increased our ability to interrogate the cellular composition of traditionally difficult to study organs, such as the pancreas. With the advent of these technologies and approaches, the field has grown, in just a few years, from profiling pancreas disease states to identifying molecular mechanisms of therapy resistance in pancreatic ductal adenocarcinoma, a particularly deadly cancer. Single-cell transcriptomics and related spatial approaches have identified previously undescribed epithelial and stromal cell types and states, how these populations change with disease progression, and potential mechanisms of action which will serve as the basis for designing new therapeutic strategies. Here, we review the recent literature on how single-cell transcriptomic approaches have changed our understanding of pancreas biology and disease progression.

Cell-Autonomous Cxcl1 Sustains Tolerogenic Circuitries and Stromal Inflammation via Neutrophil-Derived TNF in Pancreatic Cancer.

We have shown that KRAS-TP53 genomic coalteration is associated with immune-excluded microenvironments, chemoresistance, and poor survival in pancreatic ductal adenocarcinoma (PDAC) patients. By treating KRAS-TP53 cooperativity as a model for high-risk biology, we now identify cell-autonomous Cxcl1 as a key mediator of spatial T-cell restriction via interactions with CXCR2+ neutrophilic myeloid-derived suppressor cells in human PDAC using imaging mass cytometry. Silencing of cell-intrinsic Cxcl1 in LSL-KrasG12D/+;Trp53R172H/+;Pdx-1Cre/+(KPC) cells reprograms the trafficking and functional dynamics of neutrophils to overcome T-cell exclusion and controls tumor growth in a T cell-dependent manner. Mechanistically, neutrophil-derived TNF is a central regulator of this immunologic rewiring, instigating feed-forward Cxcl1 overproduction from tumor cells and cancer-associated fibroblasts (CAF), T-cell dysfunction, and inflammatory CAF polarization via transmembrane TNF-TNFR2 interactions. TNFR2 inhibition disrupts this circuitry and improves sensitivity to chemotherapy in vivo. Our results uncover cancer cell-neutrophil cross-talk in which context-dependent TNF signaling amplifies stromal inflammation and immune tolerance to promote therapeutic resistance in PDAC. SIGNIFICANCE: By decoding connections between high-risk tumor genotypes, cell-autonomous inflammatory programs, and myeloid-enriched/T cell-excluded contexts, we identify a novel role for neutrophil-derived TNF in sustaining immunosuppression and stromal inflammation in pancreatic tumor microenvironments. This work offers a conceptual framework by which targeting context-dependent TNF signaling may overcome hallmarks of chemoresistance in pancreatic cancer. This article is highlighted in the In This Issue feature, p. 1275.

Treatment-associated remodeling of the pancreatic cancer endothelium at single-cell resolution.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most treatment refractory and lethal malignancies. The diversity of endothelial cell (EC) lineages in the tumor microenvironment (TME) impacts the efficacy of antineoplastic therapies, which in turn remodel EC states and distributions. Here, we present a single-cell resolution framework of diverse EC lineages in the PDAC TME in the context of neoadjuvant chemotherapy, radiotherapy, and losartan. We analyzed a custom single-nucleus RNA-seq dataset derived from 37 primary PDAC specimens (18 untreated, 14 neoadjuvant FOLFIRINOX + chemoradiotherapy, 5 neoadjuvant FOLFIRINOX + chemoradiotherapy + losartan). A single-nucleus transcriptome analysis of 15,185 EC profiles revealed two state programs (ribosomal, cycling), four lineage programs (capillary, arterial, venous, lymphatic), and one program that did not overlap significantly with prior signatures but was enriched in pathways involved in vasculogenesis, stem-like state, response to wounding and hypoxia, and endothelial-to-mesenchymal transition (reactive EndMT). A bulk transcriptome analysis of two independent cohorts (n = 269 patients) revealed that the lymphatic and reactive EndMT lineage programs were significantly associated with poor clinical outcomes. While losartan and proton therapy were associated with reduced lymphatic ECs, these therapies also correlated with an increase in reactive EndMT. Thus, the development and inclusion of EndMT-inhibiting drugs (e.g., nintedanib) to a neoadjuvant chemoradiotherapy regimen featuring losartan and/or proton therapy may be most effective in depleting both lymphatic and reactive EndMT populations and potentially improving patient outcomes.

Genomic alterations predictive of poor clinical outcomes in pan-cancer.

BACKGROUND: Genomic alterations are highly frequent across cancers, but their prognostic impact is not well characterized in pan-cancer cohorts. Here, we use pan-cancer cohorts from TCGA and MSK-IMPACT to evaluate the associations of common genomic alterations with poor clinical outcome. MATERIALS AND METHODS: Genomic alterations in commonly altered genes were extracted from Pan-Cancer TCGA and MSK-IMPACT cohorts. Multivariable Cox regression analyses stratified by cancer type defined adjusted hazard ratios (AHRs) for disease-specific survival (DSS), progression-free survival (PFS) and overall survival (OS). RESULTS: Using TCGA we identified 32 mutated genes, and 15 copy number (CN) genes with frequency >= 4% in 9,104 patients across 28 cancers. On UVA, having a TP53-mutations or any mutation in the 31 genes (mut31) were associated with worse PFS (HR: 1.22, p < 0.0001 and HR: 1.1, p = 0.04, respectively) and DSS (HR: 1.38, p < 0.0001, and HR: 1.16, p = 0.03, respectively). CDKN2A, PTEN deletions, and MYC-amplifications were associated with PFS and DSS (p < 0.05 for all). On MVA, including TP53-mutations, mut31, CDKN2A-deletion, PTEN-deletion, and MYC-amplification, all five alterations were independently prognostic of poor PFS and DSS. Similar results were observed in an independent cohort from MSK-IMPACT (n = 7,051) where TP53 was associated with poor OS independent of mut31 and CN alterations in CDKN2A, PTEN, and MYC in primary tumors (p < 0.0001). CONCLUSIONS: TP53-mutations, CDKN2A-deletion, PTEN-deletion, and MYC-amplification are independent pan-cancer prognostic genomic alterations.

Single-nucleus and spatial transcriptome profiling of pancreatic cancer identifies multicellular dynamics associated with neoadjuvant treatment.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and treatment-refractory cancer. Molecular stratification in pancreatic cancer remains rudimentary and does not yet inform clinical management or therapeutic development. Here, we construct a high-resolution molecular landscape of the cellular subtypes and spatial communities that compose PDAC using single-nucleus RNA sequencing and whole-transcriptome digital spatial profiling (DSP) of 43 primary PDAC tumor specimens that either received neoadjuvant therapy or were treatment naive. We uncovered recurrent expression programs across malignant cells and fibroblasts, including a newly identified neural-like progenitor malignant cell program that was enriched after chemotherapy and radiotherapy and associated with poor prognosis in independent cohorts. Integrating spatial and cellular profiles revealed three multicellular communities with distinct contributions from malignant, fibroblast and immune subtypes: classical, squamoid-basaloid and treatment enriched. Our refined molecular and cellular taxonomy can provide a framework for stratification in clinical trials and serve as a roadmap for therapeutic targeting of specific cellular phenotypes and multicellular interactions.

DNA repair and immune checkpoint blockade response.

Immune checkpoint blockade (ICB) has shown immense promise for treating patients with various cancer types, but its effectiveness relies on our ability to identify likely responders. Here, we examined the association between mutations in 25 core DNA repair genes and ICB outcomes in 6619 patients across 9 cancer types with advanced disease and MSK-IMPACT tumor sequencing. Notably, we observed that mutations in 7 of the DNA repair genes (ATM, ATR, POLE, ERCC4, NBN, RAD50, PARP1) were significantly associated with improved overall survival in ICB-treated patients (p < 0.05 for all) and had significant interaction with treatment (pinteraction <0.05 for all). Similarly, DNA repair mutations were enriched in other cancer types not previously assessed and primary tumors of unknown origins, suggesting that mutations could serve as a biomarker independent of cancer type. Although our cohort was enriched in certain cancer types, such as melanoma and non-small cell lung cancer, and clinically matched samples were not assessed, our study provides a robust approach in characterizing clinically-adoptable biomarkers that can select for potential ICB responders.

Therapeutic avenues for cancer neuroscience: translational frontiers and clinical opportunities.

With increasing attention on the essential roles of the tumour microenvironment in recent years, the nervous system has emerged as a novel and crucial facilitator of cancer growth. In this Review, we describe the foundational, translational, and clinical advances illustrating how nerves contribute to tumour proliferation, stress adaptation, immunomodulation, metastasis, electrical hyperactivity and seizures, and neuropathic pain. Collectively, this expanding knowledge base reveals multiple therapeutic avenues for cancer neuroscience that warrant further exploration in clinical studies. We discuss the available clinical data, including ongoing trials investigating novel agents targeting the tumour-nerve axis, and the therapeutic potential for repurposing existing neuroactive drugs as an anti-cancer approach, particularly in combination with established treatment regimens. Lastly, we discuss the clinical challenges of these treatment strategies and highlight unanswered questions and future directions in the burgeoning field of cancer neuroscience.

Conventional type I dendritic cells maintain a reservoir of proliferative tumor-antigen specific TCF-1+ CD8+ T cells in tumor-draining lymph nodes.

In tumors, a subset of CD8+ T cells expressing the transcription factor TCF-1 drives the response to immune checkpoint blockade. We examined the mechanisms that maintain these cells in an autochthonous model of lung adenocarcinoma. Longitudinal sampling and single-cell sequencing of tumor-antigen specific TCF-1+ CD8+ T cells revealed that while intratumoral TCF-1+ CD8+ T cells acquired dysfunctional features and decreased in number as tumors progressed, TCF-1+ CD8+ T cell frequency in the tumor draining LN (dLN) remained stable. Two discrete intratumoral TCF-1+ CD8+ T cell subsets developed over time-a proliferative SlamF6+ subset and a non-cycling SlamF6- subset. Blocking dLN egress decreased the frequency of intratumoral SlamF6+ TCF-1+ CD8+ T cells. Conventional type I dendritic cell (cDC1) in dLN decreased in number with tumor progression, and Flt3L+anti-CD40 treatment recovered SlamF6+ T cell frequencies and decreased tumor burden. Thus, cDC1s in tumor dLN maintain a reservoir of TCF-1+ CD8+ T cells and their decrease contributes to failed anti-tumor immunity.

Antigen dominance hierarchies shape TCF1+ progenitor CD8 T cell phenotypes in tumors.

CD8 T cell responses against different tumor neoantigens occur simultaneously, yet little is known about the interplay between responses and its impact on T cell function and tumor control. In mouse lung adenocarcinoma, we found that immunodominance is established in tumors, wherein CD8 T cell expansion is predominantly driven by the antigen that most stably binds MHC. T cells responding to subdominant antigens were enriched for a TCF1+ progenitor phenotype correlated with response to immune checkpoint blockade (ICB) therapy. However, the subdominant T cell response did not preferentially benefit from ICB due to a dysfunctional subset of TCF1+ cells marked by CCR6 and Tc17 differentiation. Analysis of human samples and sequencing datasets revealed that CCR6+ TCF1+ cells exist across human cancers and are not correlated with ICB response. Vaccination eliminated CCR6+ TCF1+ cells and dramatically improved the subdominant response, highlighting a strategy to optimally engage concurrent neoantigen responses against tumors.

Novel genomic signature predictive of response to immune checkpoint blockade: A pan-cancer analysis from project Genomics Evidence Neo-plasia Information Exchange (GENIE).

BACKGROUND: High tumor mutation burden (TMB) and total mutation count (TMC) can be predictive of better response to immune checkpoint blockade (ICB). Nevertheless, TMB and TMC are limited by variation across cancers and inconsistent definitions due to different profiling methods (targeted vs whole genome sequencing). Our objective was to identify genomic alterations (GAs) associated with ICB response and builds a novel genomic signature predictive of ICB response, independent of TMB/TMC. METHODS: This was a pan-cancer next generation sequencing (NGS)-association study using January 2014-May 2016 data from AACR Project Genomics Evidence Neo-plasia Information Exchange (GENIE). Participants included 6619 patients with metastatic or un-resectable cancer across 9 cancer types (including 1572 ICB-treated patients). GA data was collected using next-generation sequencing (NGS) assays and downloaded from cbioportal.org. Predictive analyses for ICB response were performed to develop the signature (ImmGA). RESULTS: GAs in 16 genes were associated with improved OS in ICB-treated patients (p < 0.005). 13 GAs were associated with an OS benefit in ICB-treated patients (Pinteraction < 0.05); these genes composed the ImmGA signature. High ImmGA score (≥2 alterations out of 13 predictive GAs) was associated with better OS in ICB-treated patients (AHR:0.67, 95%CI [0.6-0.75], p = 1.4e-12), even after accounting for TMC (Pinteraction = 8e-16). High ImmGA was associated with better OS in ICB-treated patients across most cancers and across different ICB treatment modalities. CONCLUSION: A novel signature predictive of ICB response (ImmGA) was developed from 13 GAs. Further investigation of the utility of ImmGA for treatment and trial selection is warranted.

The CD155/TIGIT axis promotes and maintains immune evasion in neoantigen-expressing pancreatic cancer.

The CD155/TIGIT axis can be co-opted during immune evasion in chronic viral infections and cancer. Pancreatic adenocarcinoma (PDAC) is a highly lethal malignancy, and immune-based strategies to combat this disease have been largely unsuccessful to date. We corroborate prior reports that a substantial portion of PDAC harbors predicted high-affinity MHC class I-restricted neoepitopes and extend these findings to advanced/metastatic disease. Using multiple preclinical models of neoantigen-expressing PDAC, we demonstrate that intratumoral neoantigen-specific CD8+ T cells adopt multiple states of dysfunction, resembling those in tumor-infiltrating lymphocytes of PDAC patients. Mechanistically, genetic and/or pharmacologic modulation of the CD155/TIGIT axis was sufficient to promote immune evasion in autochthonous neoantigen-expressing PDAC. Finally, we demonstrate that the CD155/TIGIT axis is critical in maintaining immune evasion in PDAC and uncover a combination immunotherapy (TIGIT/PD-1 co-blockade plus CD40 agonism) that elicits profound anti-tumor responses in preclinical models, now poised for clinical evaluation.

Refining the Molecular Framework for Pancreatic Cancer with Single-cell and Spatial Technologies.

Pancreatic ductal adenocarcinoma (PDAC) is a treatment-refractory malignancy in urgent need of a molecular framework for guiding therapeutic strategies. Bulk transcriptomic efforts over the past decade have yielded two broad consensus subtypes: classical pancreatic/epithelial versus basal-like/squamous/quasi-mesenchymal. Although this binary classification enables prognostic stratification, it does not currently inform the administration of treatments uniquely sensitive to either subtype. Furthermore, bulk mRNA studies are challenged by distinguishing contributions from the neoplastic compartment versus other cell types in the microenvironment, which is accentuated in PDAC given that neoplastic cellularity can be low. The application of single-cell transcriptomics to pancreatic tumors has generally lagged behind other cancer types due in part to the difficulty of extracting high-quality RNA from enzymatically degradative tissue, but emerging studies have and will continue to shed light on intratumoral heterogeneity, malignant-stromal interactions, and subtle transcriptional programs previously obscured at the bulk level. In conjunction with insights provided by single-cell/nucleus dissociative techniques, spatially resolved technologies should also facilitate the contextualization of gene programs and inferred cell-cell interactions within the tumor architecture. Finally, given that patients often receive neoadjuvant chemotherapy and/or chemoradiotherapy even in resectable disease, deciphering the gene programs enriched in or induced by cytotoxic therapy will be crucial for developing insights into complementary treatments aimed at eradicating residual cancer cells. Taken together, single-cell and spatial technologies provide an unprecedented opportunity to refine the foundations laid by prior bulk molecular studies and significantly augment precision oncology efforts in pancreatic cancer.