Laser Capture Microdissection Research Articles

Picosecond laser capture microdissection based on edge catapulting combined with dielectrophoretic force

Picosecond laser capture microdissection based on edge catapulting combined with dielectrophoretic force

P28 Data-independent acquisition mass spectrometry improves spatially resolved analysis of the human skin proteome

Abstract Introduction and aims Proteomic analysis of the extracellular matrix (ECM) presents challenges because of the highly crosslinked and low-solubility nature of ECM proteins. Traditional homogenization and protein digestion approaches result in the loss of crucial information regarding protein localization and spatial relationships. To address this, spatially resolved proteomics emerges as a powerful tool for exploring heterogeneity within bulk tissues. This study aims to determine the minimum tissue volume required for comprehensive proteome coverage using data-independent acquisition mass spectrometry (DIA-MS) on skin tissue. The study focused on optimizing spatially resolved proteomic techniques to enhance depth-of-analysis while preserving spatial specificity. Methods Human abdominal skin biopsies were obtained from a single individual and subsequently cryosectioned. Histological assessment was performed through haematoxylin and eosin staining for visualization purposes. Laser-capture microdissection coupled with mass spectrometry facilitated the precise isolation of target regions. Comparative analyses were performed between data-dependent acquisition mass spectrometry (DDA-MS) and DIA-MS, with a particular emphasis on ECM proteins within the dermis. Results Our findings revealed an improvement in proteome coverage with DIA-MS compared with DDA-MS, in addition to clear scaling relationships between the depth-of-analysis and sample concentration. The Results demonstrated the superiority of DIA-MS in achieving robust and comprehensive proteomic profiles, even with minimal tissue volumes. Preliminary findings suggest the capability of DIA-MS in elucidating the complexities of the skin proteome with spatial resolution. Conclusions In conclusion, our study highlights the efficacy of DIA-MS in spatially resolved proteomics on skin tissue. The optimized approach presented here offers a reliable and efficient method for obtaining in-depth proteome information with minimal tissue requirements. These Results form the foundation for ongoing experiments, utilizing DIA-MS to advance spatially resolved proteomic analyses of human skin.

Laser axial scanning microdissection for high-efficiency dissection from uneven biological samples

Fast and efficient separation of target samples is crucial for the application of laser-assisted microdissection in the molecular biology research field. Herein, we developed a laser axial scanning microdissection (LASM) system with an 8.6 times extended depth of focus by using an electrically tunable lens. We showed that the ablation quality of silicon wafers at different depths became homogenous after using our system. More importantly, for those uneven biological tissue sections within a height difference of no more than 19.2 µm, we have demonstrated that the targets with a size of microns at arbitrary positions can be dissected efficiently without additional focusing and dissection operations. Besides, dissection experiments on various biological samples with different embedding methods, which were widely adopted in biological experiments, also have shown the feasibility of our system.

Proteogenomic characterization of difficult-to-treat breast cancer with tumor cells enriched through laser microdissection

BackgroundBreast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer death among women globally. Despite advances, there is considerable variation in clinical outcomes for patients with non-luminal A tumors, classified as difficult-to-treat breast cancers (DTBC). This study aims to delineate the proteogenomic landscape of DTBC tumors compared to luminal A (LumA) tumors.MethodsWe retrospectively collected a total of 117 untreated primary breast tumor specimens, focusing on DTBC subtypes. Breast tumors were processed by laser microdissection (LMD) to enrich tumor cells. DNA, RNA, and protein were simultaneously extracted from each tumor preparation, followed by whole genome sequencing, paired-end RNA sequencing, global proteomics and phosphoproteomics. Differential feature analysis, pathway analysis and survival analysis were performed to better understand DTBC and investigate biomarkers.ResultsWe observed distinct variations in gene mutations, structural variations, and chromosomal alterations between DTBC and LumA breast tumors. DTBC tumors predominantly had more mutations in TP53, PLXNB3, Zinc finger genes, and fewer mutations in SDC2, CDH1, PIK3CA, SVIL, and PTEN. Notably, Cytoband 1q21, which contains numerous cell proliferation-related genes, was significantly amplified in the DTBC tumors. LMD successfully minimized stromal components and increased RNA–protein concordance, as evidenced by stromal score comparisons and proteomic analysis. Distinct DTBC and LumA-enriched clusters were observed by proteomic and phosphoproteomic clustering analysis, some with survival differences. Phosphoproteomics identified two distinct phosphoproteomic profiles for high relapse-risk and low relapse-risk basal-like tumors, involving several genes known to be associated with breast cancer oncogenesis and progression, including KIAA1522, DCK, FOXO3, MYO9B, ARID1A, EPRS, ZC3HAV1, and RBM14. Lastly, an integrated pathway analysis of multi-omics data highlighted a robust enrichment of proliferation pathways in DTBC tumors.ConclusionsThis study provides an integrated proteogenomic characterization of DTBC vs LumA with tumor cells enriched through laser microdissection. We identified many common features of DTBC tumors and the phosphopeptides that could serve as potential biomarkers for high/low relapse-risk basal-like BC and possibly guide treatment selections.

Key aspects of papillomavirus infection influence the host cervicovaginal microbiome in a preclinical murine papillomavirus (MmuPV1) infection model.

Human papillomaviruses (HPVs) are the most common sexually transmitted infection in the United States and are a major etiological agent of cancers in the anogenital tract and oral cavity. Growing evidence suggests changes in the host microbiome are associated with the natural history and ultimate outcome of HPV infection. We sought to define changes in the host cervicovaginal microbiome during papillomavirus infection, persistence, and pathogenesis using the murine papillomavirus (MmuPV1) cervicovaginal infection model. Cervicovaginal lavages were performed over a time course of MmuPV1 infection in immunocompetent female FVB/N mice and extracted DNA was analyzed by qPCR to track MmuPV1 viral copy number. 16S ribosomal RNA (rRNA) gene sequencing was used to determine the composition and diversity of microbial communities throughout this time course. We also sought to determine whether specific microbial communities exist across the spectrum of MmuPV1-induced neoplastic disease. We, therefore, performed laser-capture microdissection to isolate regions of disease representing all stages of neoplastic disease progression (normal, low- and high-grade dysplasia, and cancer) from female reproductive tract tissue sections from MmuPV1-infected mice and performed 16S rRNA sequencing. Consistent with other studies, we found that the natural murine cervicovaginal microbiome is highly variable across different experiments. Despite these differences in initial microbiome composition between experiments, we observed that MmuPV1 persistence, viral load, and severity of disease influenced the composition of the cervicovaginal microbiome. These studies demonstrate that papillomavirus infection can alter the cervicovaginal microbiome.IMPORTANCEHuman papillomaviruses (HPVs) are the most common sexually transmitted infection in the United States. A subset of HPVs that infect the anogenital tract (cervix, vagina, anus) and oral cavity cause at least 5% of cancers worldwide. Recent evidence indicates that the community of microbial organisms present in the human cervix and vagina, known as the cervicovaginal microbiome, plays a role in HPV-induced cervical cancer. However, the mechanisms underlying this interplay are not well-defined. In this study, we infected the female reproductive tract of mice with a murine papillomavirus (MmuPV1) and found that key aspects of papillomavirus infection and disease influence the host cervicovaginal microbiome. This is the first study to define changes in the host microbiome associated with MmuPV1 infection in a preclinical animal model of HPV-induced cervical cancer. These results pave the way for using MmuPV1 infection models to further investigate the interactions between papillomaviruses and the host microbiome.

Somatic PDGFRB activating variants promote smooth muscle cell phenotype modulation in intracranial fusiform aneurysm

BackgroundThe fusiform aneurysm is a nonsaccular dilatation affecting the entire vessel wall over a short distance. Although PDGFRB somatic variants have been identified in fusiform intracranial aneurysms, the molecular and cellular mechanisms driving fusiform intracranial aneurysms due to PDGFRB somatic variants remain poorly understood.MethodsIn this study, single-cell sequencing and immunofluorescence were employed to investigate the phenotypic changes in smooth muscle cells within fusiform intracranial aneurysms. Whole-exome sequencing revealed the presence of PDGFRB gene mutations in fusiform intracranial aneurysms. Subsequent immunoprecipitation experiments further explored the functional alterations of these mutated PDGFRB proteins. For the common c.1684 mutation site of PDGFRβ, we established mutant smooth muscle cell lines and zebrafish models. These models allowed us to simulate the effects of PDGFRB mutations. We explored the major downstream cellular pathways affected by PDGFRBY562D mutations and evaluated the potential therapeutic effects of Ruxolitinib.ResultsSingle-cell sequencing of two fusiform intracranial aneurysms sample revealed downregulated smooth muscle cell markers and overexpression of inflammation-related markers in vascular smooth muscle cells, which was validated by immunofluorescence staining, indicating smooth muscle cell phenotype modulation is involved in fusiform aneurysm. Whole-exome sequencing was performed on seven intracranial aneurysms (six fusiform and one saccular) and PDGFRB somatic mutations were detected in four fusiform aneurysms. Laser microdissection and Sanger sequencing results indicated that the PDGFRB mutations were present in smooth muscle layer. For the c.1684 (chr5: 149505131) site mutation reported many times, further cell experiments showed that PDGFRBY562D mutations promoted inflammatory-related vascular smooth muscle cell phenotype and JAK-STAT pathway played a crucial role in the process. Notably, transfection of PDGFRBY562D in zebrafish embryos resulted in cerebral vascular anomalies. Ruxolitinib, the JAK inhibitor, could reversed the smooth muscle cells phenotype modulation in vitro and inhibit the vascular anomalies in zebrafish induced by PDGFRB mutation.ConclusionOur findings suggested that PDGFRB somatic variants played a role in regulating smooth muscle cells phenotype modulation in fusiform aneurysms and offered a potential therapeutic option for fusiform aneurysms.

Mapping dynamic molecular changes in hippocampal subregions after traumatic brain injury through spatial proteomics

BackgroundTraumatic brain injury (TBI) often results in diverse molecular responses, challenging traditional proteomic studies that measure average changes at tissue levels and fail to capture the complexity and heterogeneity of the affected tissues. Spatial proteomics offers a solution by providing insights into sub-region-specific alterations within tissues. This study focuses on the hippocampal sub-regions, analyzing proteomic expression profiles in mice at the acute (1 day) and subacute (7 days) phases of post-TBI to understand subregion-specific vulnerabilities and long-term consequences.MethodsThree mice brains were collected from each group, including Sham, 1-day post-TBI and 7-day post-TBI. Hippocampal subregions were extracted using Laser Microdissection (LMD) and subsequently analyzed by label-free quantitative proteomics.ResultsThe spatial analysis reveals region-specific protein abundance changes, highlighting the elevation of FN1, LGALS3BP, HP, and MUG-1 in the stratum moleculare (SM), suggesting potential immune cell enrichment post-TBI. Notably, established markers of chronic traumatic encephalopathy, IGHM and B2M, exhibit specific upregulation in the dentate gyrus bottom (DG2) independent of direct mechanical injury. Metabolic pathway analysis identifies disturbances in glucose and lipid metabolism, coupled with activated cholesterol synthesis pathways enriched in SM at 7-Day post-TBI and subsequently in deeper DG1 and DG2 suggesting a role in neurogenesis and the onset of recovery. Coordinated activation of neuroglia and microtubule dynamics in DG2 suggest recovery mechanisms in less affected regions. Cluster analysis revealed spatial variations post-TBI, indicative of dysregulated neuronal plasticity and neurogenesis and further predisposition to neurological disorders. TBI-induced protein upregulation (MUG-1, PZP, GFAP, TJP, STAT-1, and CD44) across hippocampal sub-regions indicates shared molecular responses and links to neurological disorders. Spatial variations were demonstrated by proteins dysregulated in both or either of the time-points exclusively in each subregion (ELAVL2, CLIC1 in PL, CD44 and MUG-1 in SM, and SHOC2, LGALS3 in DG).ConclusionsUtilizing advanced spatial proteomics techniques, the study unveils the dynamic molecular responses in distinct hippocampal subregions post-TBI. It uncovers region-specific vulnerabilities and dysregulated neuronal processes, and potential recovery-related pathways that contribute to our understanding of TBI’s neurological consequences and provides valuable insights for biomarker discovery and therapeutic targets.

Abstract PO2-16-04: Proteogenomic characterization of non-luminal A versus luminal A breast cancer tumor cells enriched by laser microdissection

Abstract Introduction: Breast cancer (BC) is classified into four widely-accepted intrinsic subtypes based on PAM (Prediction Analysis of Microarray) 50 gene expression profiles: Luminal A (LumA), Luminal B (LumB), Her2-enriched (Her2) and basal-like (Basal). Recent multi-omic studies of human BC have identified many potential therapeutic biomarkers for these subtypes and have also revealed the extensive molecular heterogeneity of the disease. Patients with LumA tumors generally have better outcomes because these tumors are typically slower-growing and responsive to hormone therapy. However, there is still considerable variation in the clinical outcomes of patients with non-LumA tumors. Here, we strive to understand the proteogenomic characteristics of intrinsically-defined non-LumA tumors in reference to LumA tumors. Methods: A total of 117 retrospectively-collected, untreated primary breast tumor specimens, with a focus on non-LumA subtypes, were selected from the Clinical Breast Care Project and consented using a HIPAA-compliant, IRB-approved protocol. The study cohort had a median patient follow-up of 9.3 years, enabling clinical outcome-related analyses. Immunohistochemistry (IHC) subtyping was used to enrich the cohort with non-LumA subtypes. Breast tumors were embedded in OCT (Optimal Cutting Temperature) compound and processed by laser microdissection (LMD) to enrich for tumor cells. DNA, RNA, and protein were simultaneously extracted from each tumor using the illustra triplePrep kit. Paired-end RNA sequencing and whole genome sequencing (WGS) were performed for 117 and 99 tumors, respectively, using the Illumina HiSeq platform. Quantitative global proteomics and phosphoproteomic analyses were performed on 112 and 50 tumors, respectively, using isobaric TMT 6-plex labeling with the “universal reference” strategy. Results: We observed significantly lower stromal, immune and microenvironment scores in non-basal-like tumors, especially the LumA subtype from LMD-processed samples, compared to that of the same tumors but bulk-processed in TCGA-breast cancer study. There was also significantly lower stromal gene expression in LMD LumA compared to TCGA LumA. Unlike a recent report on proteomics clustering of bulk-processed tumors, we did not observe a stromal-enriched cluster, probably because the use of LMD minimized stromal components. Many common patterns of somatic mutations were observed among non-LumA tumors, such as dominant TP53 mutations and 5q deletion, suggesting a potentially common cell-of-origin for non-LumA tumors, such as an ER negative cancer stem cell or progenitor cell for these tumor subtypes. Cell proliferation-associated genes were amplified and proliferation pathways strongly enriched in non-LumA tumors. In addition to this, we identified two distinct phosphoproteomic profiles for relapsed and relapse-free basal-like BC. Moreover, we also identified 17 differentially expressed phosphopeptides that could identify cases of relapsed and relapse-free basal-like BC with a significant difference in progression-free interval of surviving cases. Conclusion: This study provides an integrated proteogenomic characterization of non-LumA vs LumA tumor specimens enriched for cancer cells. We identified many common features of non-LumA tumors and also identified phosphopeptides that could serve as potential biomarkers for less aggressive basal-like BC and possibly guide treatment selections. Disclaimer: The contents of this publication are the sole responsibility of the author(s) and do not necessarily reflect the views, opinions or policies of USUHS, HJF, the DoD or the Departments of the Army, Navy or Air Force or the DOE or PNNL. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government. Citation Format: Praveen Kumar Raj Kumar, Xiaoying Lin, Tao Liu, Lori Sturtz, Marina Gritsenko, Vladislav Petyuk, Tyler Sagendorf, Brenda Deyarmin, Jianfang Liu, Anupama Praveen-Kumar, Guisong Wang, Jason McDermott, Anil Shukla, Ronald Moore, Matthew Monroe, Bobbie-Jo Webb-Robertson, Jeffrey Hooke, Leigh Fantacone-Campbell, Brad Mostoller, Leonid Kvecher, Jennifer Kane, Jennifer Melley, Stella Somiari, Patrick Soon-Shiong, Richard Smith, Richard Mural, Karin Rodland, Craig Shriver, Albert Kovatich, Hai Hu. Proteogenomic characterization of non-luminal A versus luminal A breast cancer tumor cells enriched by laser microdissection [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-16-04.

Abstract PO3-13-10: Proteogenomic characterization of primary invasive breast tumors from young women compared to matched tumors from older women

Abstract Introduction: Breast cancer (BC) in women < 40 years old accounts for ~5% of BC diagnosed in the U.S. However, young women have more aggressive tumors and worse outcomes, including higher rates of recurrence and lower disease-free and overall survival, compared to older women. To better understand mechanisms for these disparities, an integrated proteogenomic study of tumors from young (< 40) and older (≥ 60) women was performed through the Applied Proteogenomics OrganizationaL Learning and Outcomes (APOLLO) program. Methods: 34 pairs of retrospectively collected, untreated primary breast tumors from young and older women, matched by immunohistochemistry (IHC) subtype and race, were selected from the Clinical Breast Care Project. Median patient follow-up was 7 years. Tumor cells were enriched by laser microdissection and analyzed using RNA sequencing, whole genome sequencing, global proteomics, phosphoproteomics, and reverse phase protein array (RPPA). Molecular and clinical data from tumors from young and old patients in the TCGA-BC and METABRIC studies, matched by IHC subtype and race, were used to corroborate findings from our cohort. Results are presented comparing young women and their tumors to their older counterparts. Results: Young women have worse clinical outcome measured by progression-free interval. Despite matching for IHC subtypes, tumors from young women are enriched for intrinsic basal-like subtype, BRCA1/2 germline mutations, and had fewer invasive lobular BCs. Differentially enriched molecules and pathways: A number of significantly differentially expressed genes (DEGs) and proteins were detected, which were able to separate luminal subtypes of BC by patient age. IRS1 and IRS2 were enriched at both transcript and protein levels; IRS1 has been reported to promote tamoxifen resistance. We observed limited overlap of DEGs between the APOLLO, TCGA, and METABRIC datasets. In contrast, 14 of the 28 significantly upregulated Cancer Hallmark pathways were also enriched in TCGA-BC and METABRIC datasets; suggesting targeting pathways, vs genes, may be more effective for therapeutic intervention. Integrated pathway/network analysis: Integrated pathway analysis of RNA and protein levels identified 5 Hallmark pathways upregulated in younger women, including ER response and proliferative pathways, and 6 downregulated immune-response pathways; 10 of the 11 were supported by the public datasets. Immune scores were also lower. Kinase substrate enrichment analysis identified 4 kinases with increased activity in tumors from young women. ESR1/ER: We identified for the first time a significant reduction of ER protein expression while confirming previously reported lower ESR1 gene expression and copy number variations. Surprisingly, integrated pathway analysis identified elevation of early and late estrogen responses in young women. ER activation, indicated by phosphorylated ER (pS118) normalized to total ER protein, was elevated in LumA tumors from young women. Conclusions: This first-ever integrated proteogenomic study shows that BC in young women is enriched for more aggressive molecular subtypes and for genes that promote tamoxifen resistance, exhibits lower immune pathways and immune scores, and shows enhanced ER responses despite lower gene and protein expression of ER. These findings may contribute to the understanding of the molecular mechanisms underlying worse outcomes of BC in young women, and offer new insight to therapeutic strategies. Disclaimer: The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views, opinions or policies of USUHS, HJF, the DoD or the Departments of the Army, Navy or Air Force. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government. Citation Format: Praveen Kumar Raj Kumar, Jianfang Liu, Anthony Soltis, Nicholas Bateman, Qingrong Chen, Lori Sturtz, Brenda Deyarmin, Mariaelena Pierobon, Tamara Abulez, Anupama Praveen-Kumar, Xijun Zhang, Trinh Nguyen, Chunhua Yan, Ying Hu, Kate Guion, Jeffrey Hooke, Albert Kovatich, Leigh Fantacone-Campbell, Brad Mostoller, Leonid Kvecher, Stella Somiari, Patricia Steeg, Sheila Rajagopal, Kathleen Darcy, Jerry Lee, Clifton Dalgard, Thomas Conrads, Emanuel Petricoin, Daoud Meerzaman, Matthew Wilkerson, APOLLO Research Network, Xiaoying Lin, Craig Shriver, Stanley Lipkowitz, Hai Hu. Proteogenomic characterization of primary invasive breast tumors from young women compared to matched tumors from older women [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-13-10.

Abstract PO5-13-09: Clinical usage of a CLIA accredited protein/phosphoprotein assay for breast cancer: Primary usage cases and significant findings in the HER2- setting

Abstract Background: A CLIA-accredited assay that measures the expression and/or activation levels of 32 clinically actionable protein and phosphoprotein analytes in clinical tissue biopsy samples was launched in January 2021. The current intended use population for the assay is for patients with late/advanced stage breast cancer wherein molecular profiling is being utilized for clinical decision making. Assessment of physician ordering practices, clinical use dynamics as well as protein/phosphoprotein actionability frequencies were determined for clinically actionable biomarkers. Methods: A total of 219 cases have been processed to date, with mean turn-around time of 16.4 days. The assay utilizes laser capture microdissection to enrich tumor epithelium from FFPE core needle biopsy and resection material coupled to reverse phase protein array. Results for each of the 32 analytes are generated by comparing each patient to a reference population and reported both as a relative quartile and as a continuous percentile compared to the reference population (0-100%). For the purposes of this analysis, significant expression/activation cutpoints were set at the 40th percentile threshold. Results: To date, clinicians have requested the test most frequently to identify potential therapeutic targets in patients with HER2-negative disease (84% of all tested patients, 36% were triple-negative). Despite the majority of cases being HER2-negative, we found that phosphorylated (p) HER2 (Y1248) was found to be activated in 17% of all HER2-negative cases and 19% of triple negative breast cancer cases (TNBC). pHER2 was concurrently activated with pHER3 (Y1289) in 15% (12/80) of TNBC cases and 13% (24/187) of all HER2-negative cases. pHER2 presented concurrently with pEGFR (Y1068 or T654) in 14% (11/80) of TNBC cases and 14% (26/187) of all HER2- cases. Expression of TROP2 was observed in 16% of HER2-negative patients including 11% of ER+/HER2- and 23% of TNBC cancers. MHCII expression, a recently described candidate predictive biomarker of anti-PDL1/PD1 response in TNBC, was found to be expressed in 29% of TNBC patients. Activation frequencies of other clinically relevant drug targets in HER2-negative disease such AKT and mTOR pathway activation was also assessed. We found that pAKT (S473 and/or T308) was activated in 7% of ER+/HER2- cases and 14% of TNBC. Concurrent with AKT activation, at least one additional member of the AKT-mTOR pathway (mTORC1 (S2448), 4E-BP1 (S65), p70S6K (T389), or S6RP (S235/S236) was co-activated in 50% and 45% of ER+/HER2- and TNBC cancers respectively. Conclusion: This initial assessment of a CLIA LDT protein/phosphoprotein assay utilized to date found that physicians most often order the test for advanced stage HER2-negative breast cancer. Activation/expression of clinically important protein drug targets with potential actionability in HER2-negative disease was observed in a significant number of cases and demonstrates the power of the assay to elucidate actionable targets for potential therapeutic intervention. Citation Format: Claudius Mueller, Brian Corgiat, Emanuel Petricoin, Kayla Sparks, Joyce O'Shaughnessy, Chelsea Gawryletz, Matthew Schwartz, Justin Davis. Clinical usage of a CLIA accredited protein/phosphoprotein assay for breast cancer: Primary usage cases and significant findings in the HER2- setting [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-13-09.

Laser capture microdissection transcriptome (LCM RNA-seq) reveals BcDFR is a key gene in anthocyanin synthesis of non-heading Chinese cabbage

BackgroundPurple non-heading Chinese cabbage [Brassica campestris (syn. Brassica rapa) ssp. chinensis] has become popular because of its richness in anthocyanin. However, anthocyanin only accumulates in the upper epidermis of leaves. Further studies are needed to investigate the molecular mechanisms underlying the specific accumulation of it.ResultsIn this study, we used the laser capture frozen section method (LCM) to divide purple (ZBC) and green (LBC) non-heading Chinese cabbage leaves into upper and lower epidermis parts (Pup represents the purple upper epidermis, Plow represents the purple lower epidermis, Gup represents the green upper epidermis, Glow represents the green lower epidermis). Through transcriptome sequencing, we found that the DIHYDROFLAVONOL 4-REDUCTASE-encoding gene BcDFR, is strongly expressed in Pup but hardly in others (Plow, Gup, Glow). Further, a deletion and insertion in the promoter of BcDFR in LBC were found, which may interfere with BcDFR expression. Subsequent analysis of gene structure and conserved structural domains showed that BcDFR is highly conserved in Brassica species. The predicted protein-protein interaction network of BcDFR suggests that it interacts with almost all functional proteins in the anthocyanin biosynthesis pathway. Finally, the results of the tobacco transient expression also demonstrated that BcDFR promotes the synthesis and accumulation of anthocyanin.ConclusionsBcDFR is specifically highly expressed on the upper epidermis of purple non-heading Chinese cabbage leaves and regulates anthocyanin biosynthesis and accumulation. Our study provides new insights into the functional analysis and transcriptional regulatory network of anthocyanin-related genes in purple non-heading Chinese cabbage.

LMD and LC-MS-based chemical constituents and pharmacological effects assessment for two different processing methods of the root of Paeonia lactiflora Pall.

The plant of Paeonia lactiflora Pall. belongs to Ranunculaceae, and its root can be divided into two categories according to different processing methods, which included that one was directly dried without peeling the root of the P. lactiflora (PR), and the other was peeled the root of the P. lactiflora (PPR) after boiled and dried. To evaluate the difference of chemical components, UPLC-ESI-Q-Exactive Focus-MS/MS and UPLC-QQQ-MS were applied. The distribution of chemical components in different tissues was located by laser microdissection (LMD), especially the different ingredients. A total of 86 compounds were identified from PR and PPR. Four kind of tissues were isolated from the fresh root of the P. lactiflora (FPR), and 54 compounds were identified. Especially the content of gallic acid, albiflorin, and paeoniflorin with high biological activities were the highest in the cork, but they were lower in PR than that in PPR, which probably related to the process. To illustrate the difference in pharmacological effects of PR and PPR, the tonifying blood and analgesic effects on mice were investigated, and it was found that the tonifying blood and analgesic effects of PPR was superior to that of PR, even though PR had more constituents. The material basis for tonifying blood and analgesic effect of the root of P. lactiflora is likely to be associated with an increase in constituents such as paeoniflorin and paeoniflorin lactone after boiled and peeled. The study was likely to provide some theoretical support for the standard and clinical application.

Differential Gene Expression in Porcine Lung Compartments after Experimental Infection with Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (Mhyo) is the causative agent of porcine enzootic pneumonia (EP), as well as one of the main pathogens involved in the porcine respiratory disease complex. The host-pathogen interaction between Mhyo and infected pigs is complex and not completely understood; however, improving the understanding of these intricacies is essential for the development of effective control strategies of EP. In order to improve our knowledge about this interaction, laser-capture microdissection was used to collect bronchi, bronchi-associated lymphoid tissue, and lung parenchyma from animals infected with different strains of Mhyo, and mRNA expression levels of different molecules involved in Mhyo infection (ICAM1, IL-8, IL-10, IL-23, IFN-α, IFN-γ, TGF-β, and TNF-α) were analyzed by qPCR. In addition, the quantification of Mhyo load in the different lung compartments and the scoring of macroscopic and microscopic lung lesions were also performed. Strain-associated differences in virulence were observed, as well as the presence of significant differences in expression levels of cytokines among lung compartments. IL-8 and IL-10 presented the highest upregulation, with limited differences between strains and lung compartments. IFN-α was strongly downregulated in BALT, implying a relevant role for this cytokine in the immunomodulation associated with Mhyo infections. IL-23 was also upregulated in all lung compartments, suggesting the potential involvement of a Th17-mediated immune response in Mhyo infections. Our findings highlight the relevance of Th1 and Th2 immune response in cases of EP, shedding light on the gene expression levels of key cytokines in the lung of pigs at a microscopic level.

Spatial Proteomics Reveals Alcohol-Induced Damages to the Crypts and Villi of the Mouse Small Intestine.

Alcohol consumption perturbs the gut immune barrier and ultimately results in alcoholic liver diseases, but little is known about how immune-related cells in the gut are perturbed in this process. In this study, we employed laser capture microdissection and a label-free proteomics approach to investigate the consequences of alcohol exposure to the proteomes of crypts and villi in the proximal small intestine. Intestinal tissues from alcohol-fed and pair-fed mice were microdissected to selectively capture cells in the crypts and villi regions, followed by one-pot protein digestion and data-independent LC-MS/MS analysis. We successfully identified over 3000 proteins from each of the crypt or villi regions equivalent to ∼3000 cells. Analysis of alcohol-treated tissues indicated an enhanced alcohol metabolism and reduced levels of α-defensins in crypts, alongside increased lipid metabolism and apoptosis in villi. Immunofluorescence imaging further corroborated the proteomic findings. Our work provides a detailed profiling of the proteomic changes in the compartments of the mouse small intestine and aids in molecular-level understanding of alcohol-induced tissue damage.

Meta-analysis of differential gene expression in lower motor neurons isolated by laser capture microdissection from post-mortem ALS spinal cords.

ALS is a fatal neurodegenerative disease for which underlying mechanisms are incompletely understood. The motor neuron is a central player in ALS pathogenesis but different transcriptome signatures have been derived from bulk analysis of post-mortem tissue and iPSC-derived motor neurons (iPSC-MNs). This study performed a meta-analysis of six gene expression studies (microarray and RNA-seq) in which laser capture microdissection (LCM) was used to isolate lower motor neurons from post-mortem spinal cords of ALS and control (CTL) subjects. Differentially expressed genes (DEGs) with consistent ALS versus CTL expression differences across studies were identified. The analysis identified 222 ALS-increased DEGs (FDR <0.10, SMD >0.80) and 278 ALS-decreased DEGs (FDR <0.10, SMD < -0.80). ALS-increased DEGs were linked to PI3K-AKT signaling, innate immunity, inflammation, motor neuron differentiation and extracellular matrix. ALS-decreased DEGs were associated with the ubiquitin-proteosome system, microtubules, axon growth, RNA-binding proteins and synaptic membrane. ALS-decreased DEG mRNAs frequently interacted with RNA-binding proteins (e.g., FUS, HuR). The complete set of DEGs (increased and decreased) overlapped significantly with genes near ALS-associated SNP loci (p < 0.01). Transcription factor target motifs with increased proximity to ALS-increased DEGs were identified, most notably DNA elements predicted to interact with forkhead transcription factors (e.g., FOXP1) and motor neuron and pancreas homeobox 1 (MNX1). Some of these DNA elements overlie ALS-associated SNPs within known enhancers and are predicted to have genotype-dependent MNX1 interactions. DEGs were compared to those identified from SOD1-G93A mice and bulk spinal cord segments or iPSC-MNs from ALS patients. There was good correspondence with transcriptome changes from SOD1-G93A mice (r ≤ 0.408) but most DEGs were not differentially expressed in bulk spinal cords or iPSC-MNs and transcriptome-wide effect size correlations were weak (bulk tissue: r ≤ 0.207, iPSC-MN: r ≤ 0.037). This study defines a robust transcriptome signature from LCM-based motor neuron studies of post-mortem tissue from ALS and CTL subjects. This signature differs from those obtained from analysis of bulk spinal cord segments and iPSC-MNs. Results provide insight into mechanisms underlying gene dysregulation in ALS and highlight connections between these mechanisms, ALS genetics, and motor neuron biology.

Spatial omics reveals molecular changes in focal cortical dysplasia type II

Focal cortical dysplasia (FCD) represents a group of diverse localized cortical lesions that are highly epileptogenic and occur due to abnormal brain development caused by genetic mutations, involving the mammalian target of rapamycin (mTOR). These somatic mutations lead to mosaicism in the affected brain, posing challenges to unravel the direct and indirect functional consequences of these mutations. To comprehensively characterize the impact of mTOR mutations on the brain, we employed here a multimodal approach in a preclinical mouse model of FCD type II (Rheb), focusing on spatial omics techniques to define the proteomic and lipidomic changes. Mass Spectrometry Imaging (MSI) combined with fluorescence imaging and label free proteomics, revealed insight into the brain's lipidome and proteome within the FCD type II affected region in the mouse model. MSI visualized disrupted neuronal migration and differential lipid distribution including a reduction in sulfatides in the FCD type II-affected region, which play a role in brain myelination. MSI-guided laser capture microdissection (LMD) was conducted on FCD type II and control regions, followed by label free proteomics, revealing changes in myelination pathways by oligodendrocytes. Surgical resections of FCD type IIb and postmortem human cortex were analyzed by bulk transcriptomics to unravel the interplay between genetic mutations and molecular changes in FCD type II. Our comparative analysis of protein pathways and enriched Gene Ontology pathways related to myelination in the FCD type II-affected mouse model and human FCD type IIb transcriptomics highlights the animal model's translational value. This dual approach, including mouse model proteomics and human transcriptomics strengthens our understanding of the functional consequences arising from somatic mutations in FCD type II, as well as the identification of pathways that may be used as therapeutic strategies in the future.

270 Inducing Neural Maturation and Increasing Survival in SHH Medulloblastoma Using EZH2 Inhibition in a Mouse Model

INTRODUCTION: Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Genetically, MB can be divided into four subgroups of which the SHH subtype histologically shows nodular architecture. Within this nodular architecture, there are islands of mature cells, with more abundant neuropil and a low proliferation rate, scattered among sheets of primitive cells. METHODS: We performed laser capture microdissection followed by whole transcriptome analysis, spatial transcriptomics using Digital Spatial Profiling, whole genome DNA methylation and ChIP-Seq analysis of mature and primitive areas from 8 medulloblastomas. We developed a genetically-engineered mouse model of SHH MB showing spontaneous maturation and lack of maturation with a conditional EZH2 genetic ablation or EZH2 overactivation respectively. Finally, we developed a fucoidan-based nanoparticle drug delivery across the blood brain barrier (BBB) for targeted molecular inhibition. RESULTS: Using whole transcriptome and DNA methylation analysis, we identified ∼120 differentially expressed genes between primitive and mature regions with enrichment for genes regulated by H3K4me3 and H3K27me3. ChIP-Seq analysis showed striking differences in H3K27me3 enrichment between primitive and mature medulloblastoma cells including at the EZH2 locus. Medulloblastoma specific EZH2 genetic ablation resulted in diffuse tumor cell differentiation and prolonged survival in mice (n = 10 per group, log-rank p = 0.01). Conversely, conditional EZH2 (Y641F) activation prevented medulloblastoma differentiation. A fucoidan-based nanoparticle successfully delivered the EZH2 inhibitor (EPZ-6438) across the murine BBB to achieve significant extension of mouse survival (median 70 days compared to 21 days in control mice; *p = 0.01, Mantel-Cox). CONCLUSIONS: Spontaneous maturation of medulloblastoma cells can be induced by inhibition of EZH2. Fucoidan-based nanoparticle delivery systems allows tumor specific targeted delivery across the BBB extending survival in mice.

Deceptive learning in histopathology.

Deep learning holds immense potential for histopathology, automating tasks that are simple for expert pathologists and revealing novel biology for tasks that were previously considered difficult or impossible to solve by eye alone. However, the extent to which the visual strategies learned by deep learning models in histopathological analysis are trustworthy or not has yet to be systematically analysed. Here, we systematically evaluate deep neural networks (DNNs) trained for histopathological analysis in order to understand if their learned strategies are trustworthy or deceptive. We trained a variety of DNNs on a novel data set of 221 whole-slide images (WSIs) from lung adenocarcinoma patients, and evaluated their effectiveness at (1) molecular profiling of KRAS versus EGFR mutations, (2) determining the primary tissue of a tumour and (3) tumour detection. While DNNs achieved above-chance performance on molecular profiling, they did so by exploiting correlations between histological subtypes and mutations, and failed to generalise to a challenging test set obtained through laser capture microdissection (LCM). In contrast, DNNs learned robust and trustworthy strategies for determining the primary tissue of a tumour as well as detecting and localising tumours in tissue. Our work demonstrates that DNNs hold immense promise for aiding pathologists in analysing tissue. However, they are also capable of achieving seemingly strong performance by learning deceptive strategies that leverage spurious correlations, and are ultimately unsuitable for research or clinical work. The framework we propose for model evaluation and interpretation is an important step towards developing reliable automated systems for histopathological analysis.

The Mechanism of Ovule Abortion in Self-Pollinated 'Hanfu' Apple Fruits and Related Gene Screening.

Apples exhibit S-RNase-mediated self-incompatibility and typically require cross-pollination in nature. 'Hanfu' is a cultivar that produces abundant fruit after self-pollination, although it also shows a high rate of seed abortion afterwards, which greatly reduces fruit quality. In this study, we investigated the ovule development process and the mechanism of ovule abortion in apples after self-pollination. Using a DIC microscope and biomicroscope, we found that the abortion of apple ovules occurs before embryo formation and results from the failure of sperm-egg fusion. Further, we used laser-assisted microdissection (LAM) cutting and sperm and egg cell sequencing at different periods after pollination to obtain the genes related to ovule abortion. The top 40 differentially expressed genes (DEGs) were further verified, and the results were consistent with switching the mechanism at the 5' end of the RNA transcript (SMART-seq). Through this study, we can preliminarily clarify the mechanism of ovule abortion in self-pollinated apple fruits and provide a gene reserve for further study and improvement of 'Hanfu' apple fruit quality.

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

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