Disruption of cellular plasticity by repeat RNAs in human pancreatic cancer.

Aberrant expression of repeat RNAs in pancreatic ductal adenocarcinoma (PDAC) mimics viral-like responses with implications on tumor cell state and the response of the surrounding microenvironment. To better understand the relationship of repeat RNAs in human PDAC, we performed spatial molecular imaging at single-cell resolution in 46 primary tumors, revealing correlations of high repeat RNA expression with alterations in epithelial state in PDAC cells and myofibroblast phenotype in cancer-associated fibroblasts (CAFs). This loss of cellular identity is observed with dosing of extracellular vesicles (EVs) and individual repeat RNAs of PDAC and CAF cell culture models pointing to cell-cell intercommunication of these viral-like elements. Differences in PDAC and CAF responses are driven by distinct innate immune signaling through interferon regulatory factor 3 (IRF3). The cell-context-specific viral-like responses to repeat RNAs provide a mechanism for modulation of cellular plasticity in diverse cell types in the PDAC microenvironment.

HCC spatial transcriptomic profiling reveals significant and potentially targetable cancer-endothelial interactions.

BACKGROUND: HCC is a highly vascular tumor, and many effective drug regimens target the tumor blood vessels. Prior bulk HCC subtyping data used bulk transcriptomes, which contained a mixture of parenchymal and stromal contributions. METHODS: We utilized computational deconvolution and cell-cell interaction analyses to cell type-specific (tumor-enriched and vessel-enriched) spatial transcriptomic data collected from 41 resected HCC tissue specimens. RESULTS: We report that the prior Hoshida bulk transcriptional subtyping schema is driven largely by an endothelial fraction, show an alternative tumor-specific schema has potential prognostic value, and use spatially paired ligand-receptor analyses to identify known and novel (LGALS9 tumor-HAVCR2 vessel) signaling relationships that drive HCC biology in a subtype-specific and potentially targetable manner. CONCLUSIONS: Our study leverages spatial gene expression profiling technologies to dissect HCC heterogeneity and identify heterogeneous signaling relationships between cancer cells and their endothelial cells. Future validation and expansion of these findings may validate novel cancer-endothelial cell interactions and related drug targets.

Collagen type I PET/MRI enables evaluation of treatment response in pancreatic cancer in pre-clinical and first-in-human translational studies.

Pancreatic ductal adenocarcinoma (PDAC) is an invasive and rapidly progressive malignancy. A major challenge in patient management is the lack of a reliable imaging tool to monitor tumor response to treatment. Tumor-associated fibrosis characterized by high type I collagen is a hallmark of PDAC, and fibrosis further increases in response to neoadjuvant chemoradiotherapy (CRT). We hypothesized that molecular positron emission tomography (PET) using a type I collagen-specific imaging probe, 68Ga-CBP8 can detect and measure changes in tumor fibrosis in response to standard treatment in mouse models and patients with PDAC. Methods: We evaluated the specificity of 68Ga-CBP8 PET to tumor collagen and its ability to differentiate responders from non-responders based on the dynamic changes of fibrosis in nude mouse models of human PDAC including FOLFIRNOX-sensitive (PANC-1 and PDAC6) and FOLFIRINOX-resistant (SU.86.86). Next, we demonstrated the specificity and sensitivity of 68Ga-CBP8 to the deposited collagen in resected human PDAC and pancreas tissues. Eight male participant (49-65 y) with newly diagnosed PDAC underwent dynamic 68Ga-CBP8 PET/MRI, and five underwent follow up 68Ga-CBP8 PET/MRI after completing standard CRT. PET parameters were correlated with tumor collagen content and markers of response on histology. Results: 68Ga-CBP8 showed specific binding to PDAC compared to non-binding 68Ga-CNBP probe in two mouse models of PDAC using PET imaging and to resected human PDAC using autoradiography (P < 0.05 for all comparisons). 68Ga-CBP8 PET showed 2-fold higher tumor signal in mouse models following FOLFIRINOX treatment in PANC-1 and PDAC6 models (P < 0.01), but no significant increase after treatment in FOLFIRINOX resistant SU.86.86 model. 68Ga-CBP8 binding to resected human PDAC was significantly higher (P < 0.0001) in treated versus untreated tissue. PET/MRI of PDAC patients prior to CRT showed significantly higher 68Ga-CBP8 uptake in tumor compared to pancreas (SUVmean: 2.35 ± 0.36 vs. 1.99 ± 0.25, P = 0.036, n = 8). PET tumor values significantly increased following CRT compared to untreated tumors (SUVmean: 2.83 ± 0.30 vs. 2.25 ± 0.41, P = 0.01, n = 5). Collagen deposition significantly increased in response to CRT (59 ± 9% vs. 30 ± 9%, P=0.0005 in treated vs. untreated tumors). Tumor and pancreas collagen content showed a positive direct correlation with SUVmean (R2 = 0.54, P = 0.0007). Conclusions: This study demonstrates the specificity of 68Ga-CBP8 PET to tumor type I collagen and its ability to differentiate responders from non-responders based on the dynamic changes of fibrosis in PDAC. The results highlight the potential use of collagen PET as a non-invasive tool for monitoring response to treatment in patients with PDAC.

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.

ß2-microglobulin expression is associated with aggressive histology, activated tumor immune milieu, and outcome in colon carcinoma.

OBJECTIVES: We sought to assess the expression of human leukocyte antigen (HLA) proteins and ß2-microglobulin (B2M) in tumor cells and the relationship with immune microenvironment and outcome in colorectal cancer (CRC). METHODS: A total of 953 CRC cases were evaluated by immunohistochemistry for HLA class I, HLA class II, and B2M. The expression level of these biomarkers was correlated with clinicopathologic information, BRAF V600E and mismatch repair (MMR) proteins, and the quantitated expression levels of immune cells (CD8 and CD163) and immune regulatory proteins (FoxP3, programmed cell death 1 ligand 1 [PD-L1], and LAG3). RESULTS: We found that B2M-low tumors were statistically correlated with aggressive histologic features, including higher stage, higher grade, extramural venous invasion, perineural invasion, and distant metastasis. Expression of B2M was positively correlated (R2 = 0.3) and significantly associated with MMR-deficient tumors (P < .001); B2M-low tumors were also associated with an "immune cold"' microenvironment, including a reduced number of immune cells (CD8 and CD163), reduced expression of immune regulatory proteins by immune cells (PD-L1, FoxP3, and LAG3), and reduced tumor cell expression of PD-L1. These B2M-low tumors correlated with lower disease-specific survival (P = .018), a finding that maintained significance only for the proficient MMR cohort (P = .037). CONCLUSIONS: Our findings suggest that B2M expression may support predictive models for both outcome and checkpoint inhibitor therapy treatment response for colorectal adenocarcinoma.

Virtual Scribes and Physician Time Spent on Electronic Health Records.

Importance: Time on the electronic health record (EHR) is associated with burnout among physicians. Newer virtual scribe models, which enable support from either a real-time or asynchronous scribe, have the potential to reduce the burden of the EHR and EHR-related documentation. Objective: To characterize the association of use of virtual scribes with changes in physicians' EHR time and note and order composition and to identify the physician, scribe, and scribe response factors associated with changes in EHR time upon virtual scribe use. Design, Setting, and Participants: Retrospective, pre-post quality improvement study of 144 physicians across specialties who had used a scribe for at least 3 months from January 2020 to September 2022, were affiliated with Brigham and Women's Hospital and Massachusetts General Hospital, and cared for patients in the outpatient setting. Data were analyzed from November 2022 to January 2024. Exposure: Use of either a real-time or asynchronous virtual scribe. Main Outcomes: Total EHR time, time on notes, and pajama time (5:30 pm to 7:00 am on weekdays and nonscheduled weekends and holidays), all per appointment; proportion of the note written by the physician and team contribution to orders. Results: The main study sample included 144 unique physicians who had used a virtual scribe for at least 3 months in 152 unique scribe participation episodes (134 [88.2%] had used an asynchronous scribe service). Nearly two-thirds of the physicians (91 physicians [63.2%]) were female and more than half (86 physicians [59.7%]) were in primary care specialties. Use of a virtual scribe was associated with significant decreases in total EHR time per appointment (mean [SD] of 5.6 [16.4] minutes; P < .001) in the 3 months after vs the 3 months prior to scribe use. Scribe use was also associated with significant decreases in note time per appointment and pajama time per appointment (mean [SD] of 1.3 [3.3] minutes; P < .001 and 1.1 [4.0] minutes; P = .004). In a multivariable linear regression model, the following factors were associated with significant decreases in total EHR time per appointment with a scribe use at 3 months: practicing in a medical specialty (-7.8; 95% CI, -13.4 to -2.2 minutes), greater baseline EHR time per appointment (-0.3; 95% CI, -0.4 to -0.2 minutes per additional minute of baseline EHR time), and decrease in the percentage of the note contributed by the physician (-9.1; 95% CI, -17.3 to -0.8 minutes for every percentage point decrease). Conclusions and Relevance: In 2 academic medical centers, use of virtual scribes was associated with significant decreases in total EHR time, time spent on notes, and pajama time, all per appointment. Virtual scribes may be particularly effective among medical specialists and those physicians with greater baseline EHR time.

Tumor cell-based liquid biopsy using high-throughput microfluidic enrichment of entire leukapheresis product.

Circulating Tumor Cells (CTCs), interrogated by sampling blood from patients with cancer, contain multiple analytes, including intact RNA, high molecular weight DNA, proteins, and metabolic markers. However, the clinical utility of tumor cell-based liquid biopsy has been limited since CTCs are very rare, and current technologies cannot process the blood volumes required to isolate a sufficient number of tumor cells for in-depth assays. We previously described a high-throughput microfluidic prototype utilizing high-flow channels and amplification of cell sorting forces through magnetic lenses. Here, we apply this technology to analyze patient-derived leukapheresis products, interrogating a mean blood volume of 5.83 liters from patients with metastatic cancer, with a median of 2,799 CTCs purified per patient. Isolation of many CTCs from individual patients enables characterization of their morphological and molecular heterogeneity, including cell and nuclear size and RNA expression. It also allows robust detection of gene copy number variation, a definitive cancer marker with potential diagnostic applications. High-volume microfluidic enrichment of CTCs constitutes a new dimension in liquid biopsies.

Transfer Learning Reveals Cancer-Associated Fibroblasts Are Associated with Epithelial-Mesenchymal Transition and Inflammation in Cancer Cells in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by an immunosuppressive tumor microenvironment enriched with cancer-associated fibroblasts (CAF). This study used a convergence approach to identify tumor cell and CAF interactions through the integration of single-cell data from human tumors with human organoid coculture experiments. Analysis of a comprehensive atlas of PDAC single-cell RNA sequencing data indicated that CAF density is associated with increased inflammation and epithelial-mesenchymal transition (EMT) in epithelial cells. Transfer learning using transcriptional data from patient-derived organoid and CAF cocultures provided in silico validation of CAF induction of inflammatory and EMT epithelial cell states. Further experimental validation in cocultures demonstrated integrin beta 1 (ITGB1) and vascular endothelial factor A (VEGFA) interactions with neuropilin-1 mediating CAF-epithelial cell cross-talk. Together, this study introduces transfer learning from human single-cell data to organoid coculture analyses for experimental validation of discoveries of cell-cell cross-talk and identifies fibroblast-mediated regulation of EMT and inflammation. SIGNIFICANCE: Adaptation of transfer learning to relate human single-cell RNA sequencing data to organoid-CAF cocultures facilitates discovery of human pancreatic cancer intercellular interactions and uncovers cross-talk between CAFs and tumor cells through VEGFA and ITGB1.

Interplay between B7-H3 and HLA class I in the clinical course of pancreatic ductal adenocarcinoma.

Human leukocyte antigen (HLA) class I defects are associated with cancer progression. However, their prognostic significance is controversial and may be modulated by immune checkpoints. Here, we investigated whether the checkpoint B7-H3 modulates the relationship between HLA class I and pancreatic ductal adenocarcinoma (PDAC) prognosis. PDAC tumors were analyzed for the expression of B7-H3, HLA class I, HLA class II molecules, and for the presence of tumor-infiltrating immune cells. We observed defective HLA class I and HLA class II expressions in 75% and 59% of PDAC samples, respectively. HLA class I and B7-H3 expression were positively related at mRNA and protein level, potentially because of shared regulation by RELA, a sub-unit of NF-kB. High B7-H3 expression and low CD8+ T cell density were indicators of poor survival, while HLA class I was not. Defective HLA class I expression was associated with unfavorable survival only in patients with low B7-H3 expression. Favorable survival was observed only when HLA class I expression was high and B7-H3 expression low. Our results provide the rationale for targeting B7-H3 in patients with PDAC tumors displaying high HLA class I levels.

Mesothelin CAR T Cells Secreting Anti-FAP/Anti-CD3 Molecules Efficiently Target Pancreatic Adenocarcinoma and its Stroma.

PURPOSE: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells). EXPERIMENTAL DESIGN: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids. RESULTS: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors. CONCLUSIONS: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.

Customized planning of the visible maxillary arch width in harmony with interparopia width and smile width for female facial aesthetics.

BACKGROUND: The visible maxillary arch width (VAW) is an important aesthetic-determining feature. To date, there is no well-established methodology to determine the aesthetically optimal VAW in customized treatment planning. METHODS: In this study, the common traits of the dentofacial configuration were investigated in most attractive Asian and Caucasian female smiles. The smiling photo of a subject was digitally modified based on combined variations of VAW, smile width (SW), transverse facial dimensions (TFD), and vertical facial dimensions. These modified photos were assessed for aesthetics. The aesthetically essential parameters were identified, and their mathematic correlations and reference ranges were determined for different vertical facial patterns. Using the obtained results, a mathematic guidance was constructed for customized smile designing. The applicability of this guidance was tested in Asian females. RESULTS: The most attractive Asian and Caucasian female smiles have intraracial and interracial commonalities in the VAW-to-TFD ratios. The interparopia width (IPD) predominated over facial widths in determining well-matched VAW and SW. For optimal smile aesthetics, the VAW and SW were correlated as simulated by the formula 1.92 IPD ≤ VAW + 2.3 SW ≤ 2.17 IPD, plus the VAW-to-IPD ratio within 0.54 to 0.62 and the SW-to-IPD ratio within 0.61 to 0.71, ranges tailored to vertical facial patterns. This constitutes a mathematic guidance for customized planning of the aesthetically optimal VAW. This guidance was preliminarily validated to be applicable to Asian females. CONCLUSIONS: The VAW-to-TFD ratios were essential for Caucasian and Asian female smile aesthetics. The mathematic guidance could serve as a reference for customized smile designs for Asian females.

Dysregulated Repeat Element Viral-like Immune Response in Hepatocellular Carcinoma.

Purpose: Dysregulation of viral-like repeat RNAs are a common feature across many malignancies that are linked with immunological response, but the characterization of these in hepatocellular carcinoma (HCC) is understudied. In this study, we performed RNA in situ hybridization (RNA-ISH) of different repeat RNAs, immunohistochemistry (IHC) for immune cell subpopulations, and spatial transcriptomics to understand the relationship of HCC repeat expression, immune response, and clinical outcomes. Experimental Design: RNA-ISH for LINE1, HERV-K, HERV-H, and HSATII repeats and IHC for T-cell, Treg, B-cell, macrophage, and immune checkpoint markers were performed on 43 resected HCC specimens. Spatial transcriptomics on tumor and vessel regions of interest was performed on 28 specimens from the same cohort. Results: High HERV-K and high LINE1 expression were both associated with worse overall survival. There was a positive correlation between LINE1 expression and FOXP3 T-regulatory cells (r = 0.51 p < 0.001) as well as expression of the TIM3 immune checkpoint (r = 0.34, p = 0.03). Spatial transcriptomic profiling of HERV-K high and LINE-1 high tumors identified elevated expression of multiple genes previously associated with epithelial mesenchymal transition, cellular proliferation, and worse overall prognosis in HCC including SSX1, MAGEC2, and SPINK1. Conclusion: Repeat RNAs may serve as useful prognostic biomarkers in HCC and may also serve as novel therapeutic targets. Additional study is needed to understand the mechanisms by which repeat RNAs impact HCC tumorigenesis.

Design and Demonstration of a Microelectromechanical System Single-Ring Resonator with Inner Ring-Shaped Spring Supports for Inertial Sensors.

This paper presents a novel single-ring resonator design and experimentally demonstrates its dynamic behavior. The proposed ring resonator design is simple and has a solid anchor at its center connected to an outside ring via inner ring-shaped springs. The mode shapes and frequency of the ring resonator were determined numerically and compared with analytical approaches, and the minimum split frequency was observed for the n = 3 mode of vibration. Numerical and analytical methods were used to determine the resonance frequencies, pull-in voltage, resonance frequency shift and harmonic response of the ring resonator for different silicon orientations. The split frequency in the n = 3 mode of vibration increases by the applied DC bias voltage almost by the same amount for all types of silicon. When an AC voltage with a 180-degree phase is applied to two opposite electrodes, the ring has two resonance frequencies in mode n = 2, and when the AC voltage applied to two opposite electrodes is in the same phase, the ring has one resonance frequency regardless of the crystal orientation of silicon. Prototypes were fabricated using a double silicon-on-insulator-based wafer fabrication technique and were tested to verify the resonator performance.

Proceedings of the inaugural Dark Genome Symposium: November 2022.

In November 2022 the first Dark Genome Symposium was held in Boston, USA. The meeting was hosted by Rome Therapeutics and Enara Bio, two biotechnology companies working on translating our growing understanding of this vast genetic landscape into therapies for human disease. The spirit and ambition of the meeting was one of shared knowledge, looking to strengthen the network of researchers engaged in the field. The meeting opened with a welcome from Rosana Kapeller and Kevin Pojasek followed by a first session of field defining talks from key academics in the space. A series of panels, bringing together academia and industry views, were then convened covering a wide range of pertinent topics. Finally, Richard Young and David Ting gave their views on the future direction and promise for patient impact inherent in the growing understanding of the Dark Genome.

The landscape of human SVA retrotransposons.

SINE-VNTR-Alu (SVA) retrotransposons are evolutionarily young and still-active transposable elements (TEs) in the human genome. Several pathogenic SVA insertions have been identified that directly mutate host genes to cause neurodegenerative and other types of diseases. However, due to their sequence heterogeneity and complex structures as well as limitations in sequencing techniques and analysis, SVA insertions have been less well studied compared to other mobile element insertions. Here, we identified polymorphic SVA insertions from 3646 whole-genome sequencing (WGS) samples of >150 diverse populations and constructed a polymorphic SVA insertion reference catalog. Using 20 long-read samples, we also assembled reference and polymorphic SVA sequences and characterized the internal hexamer/variable-number-tandem-repeat (VNTR) expansions as well as differing SVA activity for SVA subfamilies and human populations. In addition, we developed a module to annotate both reference and polymorphic SVA copies. By characterizing the landscape of both reference and polymorphic SVA retrotransposons, our study enables more accurate genotyping of these elements and facilitate the discovery of pathogenic SVA insertions.

Spatial transcriptomics reveals distinct tissue niches linked with steroid responsiveness in acute gastrointestinal GVHD.

Severe acute graft-versus-host disease (aGVHD) is associated with significant mortality and morbidity, especially in steroid-resistant (SR) cases. Spatial transcriptomic technology can elucidate tissue-based interactions in vivo and possibly identify predictors of treatment response. Tissue sections from 32 treatment-naïve patients with biopsy-confirmed lower gastrointestinal (GI) aGVHD were obtained. The GeoMx digital spatial profiler was used to capture transcriptome profiles of >18 000 genes from different foci of immune infiltrates, colonic epithelium, and vascular endothelium. Each tissue compartment sampled showed 2 distinct clusters that were analyzed for differential expression and spatially resolved correlation of gene signatures. Classic cell-mediated immunity signatures, normal differentiated epithelial cells, and inflamed vasculature dominated foci sampled from steroid-sensitive cases. In contrast, a neutrophil predominant noncanonical inflammation with regenerative epithelial cells and some indication of angiogenic endothelial response was overrepresented in areas from SR cases. Evaluation of potential prognostic biomarkers identified ubiquitin specific peptidase 17-like (USP17L) family of genes as being differentially expressed in immune cells from patients with worsened survival. In summary, we demonstrate distinct tissue niches with unique gene expression signatures within lower GI tissue from patients with aGVHD and provide evidence of a potential prognostic biomarker.

Immune microenvironment and lymph node yield in colorectal cancer.

BACKGROUND: Lymph node (LN) harvesting is associated with outcomes in colonic cancer. We sought to interrogate whether a distinctive immune milieu of the primary tumour is associated with LN yield. METHODS: A total of 926 treatment-naive patients with colorectal adenocarcinoma with more than 12 LNs (LN-high) were compared with patients with 12 or fewer LNs (LN-low). We performed immunohistochemistry and quantification on tissue microarrays for HLA class I/II proteins, beta-2-microglobulin (B2MG), CD8, CD163, LAG3, PD-L1, FoxP3, and BRAF V600E. RESULTS: The LN-high group was comprised of younger patients, longer resections, larger tumours, right-sided location, and tumours with deficient mismatch repair (dMMR). The tumour microenvironment showed higher CD8+ cells infiltration and B2MG expression on tumour cells in the LN-high group compared to the LN-low group. The estimated mean disease-specific survival was higher in the LN-high group than LN-low group. On multivariate analysis for prognosis, LN yield, CD8+ cells, extramural venous invasion, perineural invasion, and AJCC stage were independent prognostic factors. CONCLUSION: Our findings corroborate that higher LN yield is associated with a survival benefit. LN yield is associated with an immune high microenvironment, suggesting that tumour immune milieu influences the LN yield.

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.

Cancer cells co-evolve with retrotransposons to mitigate viral mimicry.

Overexpression of repetitive elements is an emerging hallmark of human cancers 1 . Diverse repeats can mimic viruses by replicating within the cancer genome through retrotransposition, or presenting pathogen-associated molecular patterns (PAMPs) to the pattern recognition receptors (PRRs) of the innate immune system 2-5 . Yet, how specific repeats affect tumor evolution and shape the tumor immune microenvironment (TME) in a pro- or anti-tumorigenic manner remains poorly defined. Here, we integrate whole genome and total transcriptome data from a unique autopsy cohort of multiregional samples collected in pancreatic ductal adenocarcinoma (PDAC) patients, into a comprehensive evolutionary analysis. We find that more recently evolved S hort I nterspersed N uclear E lements (SINE), a family of retrotransposable repeats, are more likely to form immunostimulatory double-strand RNAs (dsRNAs). Consequently, younger SINEs are strongly co-regulated with RIG-I like receptor associated type-I interferon genes but anti-correlated with pro-tumorigenic macrophage infiltration. We discover that immunostimulatory SINE expression in tumors is regulated by either L ong I nterspersed N uclear E lements 1 (LINE1/L1) mobility or ADAR1 activity in a TP53 mutation dependent manner. Moreover, L1 retrotransposition activity tracks with tumor evolution and is associated with TP53 mutation status. Altogether, our results suggest pancreatic tumors actively evolve to modulate immunogenic SINE stress and induce pro-tumorigenic inflammation. Our integrative, evolutionary analysis therefore illustrates, for the first time, how dark matter genomic repeats enable tumors to co-evolve with the TME by actively regulating viral mimicry to their selective advantage.

Clinical, pathological, genetics and intratumoural immune milieu of micropapillary carcinoma of the colon.

AIM: Micropapillary carcinoma (MPC) is a recognised WHO variant of colonic carcinoma (CC), although little is known about its prognosis, immune microenvironment and molecular alterations. We investigated its clinical, pathological and immunological characteristics. METHODS: We assessed 903 consecutive CCs and used the WHO definition to identify MPC. We recorded serrated and mucinous differentiation and mismatch repair (MMR) status. We performed immunohistochemistry and quantification on tissue microarrays for HLA class I/II proteins, beta-2-microglobulin (B2MG), CD8, CD163, LAG3, PD-L1, FoxP3, PD-L1and BRAF V600E. RESULTS: We classified 8.6% (N=78) of CC as MPC. Relative to non-MPC, MPC was more often high grade (p=0.03) and showed serrated morphology (p<0.01); however, we found no association with extramural venous invasion (p=0.41) and American Joint Committee on Cancer stage (p=0.95). MPCs showed lower numbers of CD8 positive lymphocytes (p<0.01), lower tumour cell B2MG expression (p=0.04) and lower tumour cell PD-L1 expression (p<0.01). There was no difference in HLA class I/II, LAG3, FOXP3, CD163 and PD-L1 positive histiocytes. There was no association with MMR status or BRAF V600E relative to non-MPC. MPC was not associated with decreased disease-specific survival (p=0.36). CONCLUSION: MPCs are associated with high-grade differentiation and a less active immune microenvironment than non-MPC. MPC is not associated with inferior disease-specific survival.