SpaceANOVA: Spatial Co-occurrence Analysis of Cell Types in Multiplex Imaging Data Using Point Process and Functional ANOVA.

Multiplex imaging platforms have enabled the identification of the spatial organization of different types of cells in complex tissue or the tumor microenvironment. Exploring the potential variations in the spatial co-occurrence or colocalization of different cell types across distinct tissue or disease classes can provide significant pathological insights, paving the way for intervention strategies. However, the existing methods in this context either rely on stringent statistical assumptions or suffer from a lack of generalizability. We present a highly powerful method to study differential spatial co-occurrence of cell types across multiple tissue or disease groups, based on the theories of the Poisson point process and functional analysis of variance. Notably, the method accommodates multiple images per subject and addresses the problem of missing tissue regions, commonly encountered due to data-collection complexities. We demonstrate the superior statistical power and robustness of the method in comparison with existing approaches through realistic simulation studies. Furthermore, we apply the method to three real data sets on different diseases collected using different imaging platforms. In particular, one of these data sets reveals novel insights into the spatial characteristics of various types of colorectal adenoma.

SpaceANOVA: Spatial co-occurrence analysis of cell types in multiplex imaging data using point process and functional ANOVA.

Motivation: Multiplex imaging platforms have enabled the identification of the spatial organization of different types of cells in complex tissue or tumor microenvironment (TME). Exploring the potential variations in the spatial co-occurrence or co-localization of different cell types across distinct tissue or disease classes can provide significant pathological insights, paving the way for intervention strategies. However, the existing methods in this context either rely on stringent statistical assumptions or suffer from a lack of generalizability. Results: We present a highly powerful method to study differential spatial co-occurrence of cell types across multiple tissue or disease groups, based on the theories of the Poisson point process (PPP) and functional analysis of variance (FANOVA). Notably, the method accommodates multiple images per subject and addresses the problem of missing tissue regions, commonly encountered in such a context due to the complex nature of the data-collection procedure. We demonstrate the superior statistical power and robustness of the method in comparison to existing approaches through realistic simulation studies. Furthermore, we apply the method to three real datasets on different diseases collected using different imaging platforms. In particular, one of these datasets reveals novel insights into the spatial characteristics of various types of precursor lesions associated with colorectal cancer. Availability: The associated R package can be found here, https://github.com/sealx017/SpaceANOVA.

Targeting TACC3 represents a novel vulnerability in highly aggressive breast cancers with centrosome amplification.

Centrosome amplification (CA) is a hallmark of cancer that is strongly associated with highly aggressive disease and worse clinical outcome. Clustering extra centrosomes is a major coping mechanism required for faithful mitosis of cancer cells with CA that would otherwise undergo mitotic catastrophe and cell death. However, its underlying molecular mechanisms have not been fully described. Furthermore, little is known about the processes and players triggering aggressiveness of cells with CA beyond mitosis. Here, we identified Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3) to be overexpressed in tumors with CA, and its high expression is associated with dramatically worse clinical outcome. We demonstrated, for the first time, that TACC3 forms distinct functional interactomes regulating different processes in mitosis and interphase to ensure proliferation and survival of cancer cells with CA. Mitotic TACC3 interacts with the Kinesin Family Member C1 (KIFC1) to cluster extra centrosomes for mitotic progression, and inhibition of this interaction leads to mitotic cell death via multipolar spindle formation. Interphase TACC3 interacts with the nucleosome remodeling and deacetylase (NuRD) complex (HDAC2 and MBD2) in nucleus to inhibit the expression of key tumor suppressors (e.g., p21, p16 and APAF1) driving G1/S progression, and its inhibition blocks these interactions and causes p53-independent G1 arrest and apoptosis. Notably, inducing CA by p53 loss/mutation increases the expression of TACC3 and KIFC1 via FOXM1 and renders cancer cells highly sensitive to TACC3 inhibition. Targeting TACC3 by guide RNAs or small molecule inhibitors strongly inhibits growth of organoids and breast cancer cell line- and patient-derived xenografts with CA by induction of multipolar spindles, mitotic and G1 arrest. Altogether, our results show that TACC3 is a multifunctional driver of highly aggressive breast tumors with CA and that targeting TACC3 is a promising approach to tackle this disease.

Utilizing multimodal mass spectrometry imaging for profiling immune cell composition and N-glycosylation across colorectal carcinoma disease progression.

Colorectal cancer (CRC) stands as a leading cause of death worldwide, often arising from specific genetic mutations, progressing from pre-cancerous adenomas to adenocarcinomas. Early detection through regular screening can result in a 90% 5-year survival rate for patients. However, unfortunately, only a fraction of CRC cases are identified at pre-invasive stages, allowing progression to occur silently over 10-15 years. The intricate interplay between the immune system and tumor cells within the tumor microenvironment plays a pivotal role in the progression of CRC. Immune cell clusters can either inhibit or facilitate tumor initiation, growth, and metastasis. To gain a better understanding of this relationship, we conducted N-glycomic profiling using matrix-assisted laser desorption-ionization mass spectrometry imaging (MALDI-MSI). We detected nearly 100 N-glycan species across all samples, revealing a shift in N-glycome profiles from normal to cancerous tissues, marked by a decrease in high mannose N-glycans. Further analysis of precancerous to invasive carcinomas showed an increase in pauci-mannose biantennary, and tetraantennary N-glycans with disease progression. Moreover, a distinct stratification in the N-glycome profile was observed between non-mucinous and mucinous CRC tissues, driven by pauci-mannose, high mannose, and bisecting N-glycans. Notably, we identified immune clusters of CD20+ B cells and CD3/CD44+ T cells distinctive and predictive with signature profiles of bisecting and branched N-glycans. These spatial N-glycan profiles offer potential biomarkers and therapeutic targets throughout the progression of CRC.

Response and recurrence correlates in individuals treated with neoadjuvant anti-PD-1 therapy for resectable oral cavity squamous cell carcinoma.

Neoadjuvant PD-1 blockade may be efficacious in some individuals with high-risk, resectable oral cavity head and neck cancer. To explore correlates of response patterns to neoadjuvant nivolumab treatment and post-surgical recurrences, we analyzed longitudinal tumor and blood samples in a cohort of 12 individuals displaying 33% responsiveness. Pretreatment tumor-based detection of FLT4 mutations and PTEN signature enrichment favors response, and high tumor mutational burden improves recurrence-free survival. In contrast, preexisting and/or acquired mutations (in CDKN2A, YAP1, or JAK2) correlate with innate resistance and/or tumor recurrence. Immunologically, tumor response after therapy entails T cell receptor repertoire diversification in peripheral blood and intratumoral expansion of preexisting T cell clones. A high ratio of regulatory T to T helper 17 cells in pretreatment blood predicts low T cell receptor repertoire diversity in pretreatment blood, a low cytolytic T cell signature in pretreatment tumors, and innate resistance. Our study provides a molecular framework to advance neoadjuvant anti-PD-1 therapy for individuals with resectable head and neck cancer.

Neoadjuvant presurgical PD-1 inhibition in oral cavity squamous cell carcinoma.

Oral cavity squamous cell carcinoma (OCSCC) is a prevalent surgically treated subset of head and neck cancer with frequent recurrence and poor survival. Immunotherapy has demonstrated efficacy in recurrent/metastatic head and neck cancer. However, whether antitumor responses could be fostered by neoadjuvant presurgical immunotherapy remains unclear. Using a Simon's two-stage design, we present results of a single-arm phase-II trial where 12 patients with stage II-IVA OCSCC received 3 to 4 biweekly doses of 3 mg/kg nivolumab followed by definitive surgical resection with curative intent. Presurgical nivolumab therapy in this cohort shows an overall response rate of 33% (n = 4 patients; 95% CI: 12%-53%). With a median follow up of 2.23 years, 10 out of 12 treated patients remain alive. Neoadjuvant nivolumab is safe, well-tolerated, and is not associated with delays in definitive surgical treatment in this study. This work demonstrates feasibility and safety for incorporation of nivolumab in the neoadjuvant setting for OCSCC (ClinicalTrials.gov: NCT03021993).

Differential membrane type 1 matrix metalloproteinase substrate processing with ischemia-reperfusion: relationship to interstitial microRNA dynamics and myocardial function.

OBJECTIVES: Membrane type 1 matrix metalloproteinase (MT1-MMP) is critical to a number of proteolytic and profibrotic events. However, upstream regulation of MT1-MMP with myocardial ischemia-reperfusion remains poorly understood. MicroRNAs regulate post-transcriptional events, and in silico mapping has identified a conserved sequence in MT1-MMP for microRNA-133a. This study tested the hypothesis that changes in microRNA-133a regulation occur with myocardial ischemia-reperfusion, which contributes to time- and region-dependent changes in MT1-MMP activity and processing of MT1-MMP substrates. METHODS: Yorkshire pigs (n = 12) underwent ischemia-reperfusion (90 minutes ischemia and 120 minutes reperfusion), where regional preload recruitable stroke work (sonomicrometry), interstitial MT1-MMP activity (microdialysis), Smad2 abundance (immunoblotting), and interstitial microRNA-133a (polymerase chain reaction) were determined within the ischemia-reperfusion and remote regions. Human left ventricular fibroblasts were transduced with microRNA-133a and anti-microRNA-133a (lentivirus) to determine the effects on MT1-MMP protein abundance. RESULTS: With ischemia-reperfusion, regional preload recruitable stroke work decreased from steady state (139 ± 20 mm Hg to 44 ± 11 mm Hg, P < .05) within the ischemia-reperfusion region. MT1-MMP activity increased in both regions. Phosphorylated Smad2 increased within the ischemia-reperfusion region. Both in vitro and in vivo interstitial levels of microRNA-133a decreased with ischemia and returned to steady-state levels with reperfusion. In vitro transduction of microRNA-133a in left ventricular fibroblasts decreased MT1-MMP levels. CONCLUSIONS: Modulation of MT1-MMP activity and microRNA-133a exportation into the myocardial interstitium occurred in the setting of acute myocardial ischemia-reperfusion. In addition, changes in microRNA-133a expression in left ventricular fibroblasts resulted in an inverse modulation of MT1-MMP abundance. Therefore, targeting of microRNA-133a represents a potentially novel means for regulating the cascade of profibrotic events after ischemia-reperfusion.