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Abstract Hanging drop cultures provide a favorable environment for the gentle, gel-free formation of highly uniform three-dimensional cell cultures often used in drug screening applications. Initial cell numbers can be limited, as with primary cells provided by minimally invasive biopsies. Therefore, it can be beneficial to divide cells into miniaturized arrays of hanging drops to supply a larger number of samples. Here, we present a framework for the miniaturization of hanging drop networks to nanoliter volumes. The principles of a single hanging drop are described and used to construct the fundamental equations for a microfluidic system composed of multiple connected drops. Constitutive equations for the hanging drop as a nonlinear capacitive element are derived for application in the electronic-hydraulic analogy, forming the basis for more complex, time-dependent numerical modeling of hanging drop networks. This is supplemented by traditional computational fluid dynamics simulation to provide further information about flow conditions within the wells. A fabrication protocol is presented and demonstrated for creating transparent, microscale arrays of pinned hanging drops. A custom interface, pressure-based fluidic system, and environmental chamber have been developed to support the device. Finally, fluid flow on the chip is demonstrated to align with expected behavior based on the principles derived for hanging drop networks. Challenges with the system and potential areas for improvement are discussed. This paper expands on the limited body of hanging drop network literature and provides a framework for designing, fabricating, and operating these systems at the microscale.

Abstract Ex vivo drug screening is a potentially powerful tool for the future of cancer care, but the accuracy of results is contingent on the culture model. Both monolayer (2D) and spheroid (3D) culture systems offer advantages, but given the differences in mechanical environment, we hypothesized that that the suitability of one system over another would be critical for screening drugs with mechanical targets in mechanical tissues. HCC827 lung adenocarcinoma cells were challenged with EGFR tyrosine kinase inhibitors in monolayer and spheroid culture. RNA sequencing was performed on cells in both conditions to assess culture-induced transcriptional changes that could account for differences in drug response and differences in EGFR expression detected by immunostain. A microRNA microarray was performed to assess culture-induced differences in regulation of microRNA, and the impact of miR-146a-5p on drug response was verified by inhibition. Results were confirmed in human lung adenocarcinoma tissue. HCC827 spheroids were resistant to erlotinib and gefitinib, but significantly more sensitive in 2D culture. RNA-seq and immunostaining show a discrepancy in EGFR transcript and protein expression between the two conditions, which we attribute to miR-146a-5p. This microRNA targets EGFR and is differentially expressed between 2D and 3D culture. Inhibition of miR-146a-5p significantly increased erlotinib cytotoxicity, but validation in patient-derived spheroids suggests that the effect may be mutation-specific. Analysis of RNA-seq data suggests that cells in 2D culture become highly dependent on EGFR signaling to drive proliferation and cell spreading, resulting in a misleading level of sensitivity to EGFR TKIs, while the same cells in spheroid culture retain microRNA-driven EGFR feedback regulation that leaves them less vulnerable to EGFR inhibition. These findings underscore the need for close scrutiny of culture-induced effects on drug target regulation in model design for ex vivo drug screening.

BackgroundOnly 20 percent of renal and bladder cancer patients will show a significant response to immune checkpoint inhibitor (ICI) therapy, and no test currently available accurately predicts ICI response.MethodsWe developed an “immunotumoroid” cell model system that recapitulates the tumor, its microenvironment, and necessary immune system components in patient-derived spheroids to enable ex vivo assessment of tumor response to ICI therapy. Immunotumoroids were developed from surgically resected renal cell carcinomas and bladder carcinomas selected for high tumor-infiltrating lymphocytes (TILs) and survived more than a month without media exchange. Immunohistochemistry was used to detect immune and non-immune cells in cryopreserved source tumors and the resulting immunotumoroids. Immunotumoroid response to ICIs (nivolumab, pembrolizumab, and durvalumab) and chemotherapy (cisplatin, gemcitabine, and paclitaxel) was monitored in real-time with Cytotox Red staining in an Incucyte device, and the immunotumoroid response was compared to retrospective clinical drug responses.ResultsSix of the 13 cases tested grew viable immunotumoroid models, with failed cases attributed to extensive tumor tissue necrosis or excess lymphocytes preventing spheroid formation. One successfully cultured case was excluded from the study due to low TIL infiltration (

Bias in neural network model training datasets has been observed to decrease prediction accuracy for groups underrepresented in training data. Thus, investigating the composition of training datasets used in machine learning models with healthcare applications is vital to ensure equity. Two such machine learning models are NetMHCpan-4.1 and NetMHCIIpan-4.0, used to predict antigen binding scores to major histocompatibility complex class I and II molecules, respectively. As antigen presentation is a critical step in mounting the adaptive immune response, previous work has used these or similar predictions models in a broad array of applications, from explaining asymptomatic viral infection to cancer neoantigen prediction. However, these models have also been shown to be biased toward hydrophobic peptides, suggesting the network could also contain other sources of bias. Here, we report the composition of the networks’ training datasets are heavily biased toward European Caucasian individuals and against Asian and Pacific Islander individuals. We test the ability of NetMHCpan-4.1 and NetMHCpan-4.0 to distinguish true binders from randomly generated peptides on alleles not included in the training datasets. Unexpectedly, we fail to find evidence that the disparities in training data lead to a meaningful difference in prediction quality for alleles not present in the training data. We attempt to explain this result by mapping the HLA sequence space to determine the sequence diversity of the training dataset. Furthermore, we link the residues which have the greatest impact on NetMHCpan predictions to structural features for three alleles (HLA-A*34:01, HLA-C*04:03, HLA-DRB1*12:02).

Introduction: SCLC is an aggressive malignancy with poor outcome. Most patients have disease recurrence despite treatments with multiple modalities. Subtyping of SCLC has been proposed recently, and novel agents targeting specific subtypes are actively being investigated. In this study, we evaluated the plasticity of subtypes in paired pre- and post-treatment samples. The aim was to understand possible subtype evolution after chemotherapy resistance that could lead to alternate targeted therapy strategies. Methods: A total of 68 samples from 32 patients with sufficient paired specimens were identified from 1998 to 2022. ASCL1, NEUROD1, and POU2F3 immunohistochemistry studies were performed on all cases, and subtyping by predominant expression was determined. Subtype comparison in each patient was performed, and expression analysis was performed on the basis of subtypes. Results: Of 32 cases, 28 (88%) had the same subtype in pre- and first post-treatment specimens. Protein expression level of subtype-specific transcription factor remained stable after chemotherapy. Two of five (40%) NEUROD1-predominant SCLC switched to ASCL1-predominant phenotype after treatment. One case had a pitfall of scoring ASCL1 on specimen with marked crushing artifacts. One case revealed the challenge of proper subtyping for samples with borderline POU2F3 expression. Conclusions: Subtype of SCLC generally remains the same after acquiring chemotherapy resistance. Plasticity was observed with rare cases switching from NEUROD1-predominant to ASC1-predominant SCLC. Resubtyping is unnecessary for the consideration of novel subtype-specific targeted agents, except cases with NEUROD1-predominant subtype.

Background Immune checkpoint inhibitors (ICIs) are now a first-line treatment option for patients with pleural mesothelioma with the recent approval of ipilimumab and nivolumab. Mesothelioma has a low tumor mutation burden and no robust predictors of survival with ICI. Since ICIs enable adaptive antitumor immune responses, we investigated T-cell receptor (TCR) associations with survival in participants from two clinical trials treated with ICI.Methods We included patients with pleural mesothelioma who were treated with nivolumab (NivoMes, NCT02497508) or nivolumab and ipilimumab (INITIATE, NCT03048474) after first-line therapy. TCR sequencing was performed with the ImmunoSEQ assay in 49 and 39 pretreatment and post-treatment patient peripheral blood mononuclear cell (PBMC) samples. These data were integrated with TCR sequences found in bulk RNAseq data by TRUST4 program in 45 and 35 pretreatment and post-treatment tumor biopsy samples and TCR sequences from over 600 healthy controls. The TCR sequences were clustered into groups of shared antigen specificity using GIANA. Associations of TCR clusters with overall survival were determined by cox proportional hazard analysis.Results We identified 4.2 million and 12 thousand complementarity-determining region 3 (CDR3) sequences from PBMCs and tumors, respectively, in patients treated with ICI. These CDR3 sequences were integrated with 2.1 million publically available CDR3 sequences from healthy controls and clustered. ICI-enhanced T-cell infiltration and expanded T cell diversity in tumors. Cases with TCR clones in the top tertile in the pretreatment tissue or in circulation had significantly better survival than the bottom two tertiles (p

Major Histocompability Complex (MHC) Class I molecules allow cells to present foreign and endogenous peptides to T-Cells so that cells infected by pathogens can be identified and killed. Neural networks tools such as NetMHC-4.0 and NetMHCpan-4.1 are used to predict whether peptides will bind to variants of MHC molecules. These tools are trained on data gathered from binding affinity and eluted ligand experiments. However, these tools do not track hydrophobicity, a significant biochemical factor relevant to peptide binding, in their predictions. A previous study had concluded that the peptides predicted to bind to HLA-A*0201 by NetMHC-4.0 were much more hydrophobic than expected. This paper expands that study by also focusing on HLA-B*2705 and HLA-B*0801, which prefer binding hydrophilic and balanced peptides respectively. The correlation of hydrophobicity of 9-mer peptides with their predicted binding strengths to these various HLAs was investigated. Two studies were performed, one using the data that the two neural networks were trained on, and the other using a sample of the human proteome. NetMHC-4.0 was found to have a statistically significant bias towards predicting highly hydrophobic peptides as strong binders to HLA-A*0201 and HLA-B*2705 in both studies. Machine Learning metrics were used to identify the causes for this bias: hydrophobic false positives and hydrophilic false negatives. These results suggest that the retraining the neural networks with biochemical attributes such as hydrophobicity and better training data could increase the accuracy of their predictions. This would increase their impact in applications such as vaccine design and neoantigen identification.

Abstract This case report underlines the importance of molecular characterization of genomic duplications and other structural variants in the prenatal setting to guide clinical interpretation, genetic counseling, and perinatal medical care.

Abstract Synucleinopathies are mostly sporadic neurodegenerative disorders of partly unexplained aetiology, and include Parkinson’s disease (PD) and multiple system atrophy (MSA). We have further investigated our recent finding of somatic SNCA (α-synuclein) copy number variants (CNVs, specifically gains) in synucleinopathies, using Fluorescent in-situ Hybridisation for SNCA, and single-cell whole genome sequencing for the first time in a synucleinopathy. In the cingulate cortex, mosaicism levels for SNCA gains were higher in MSA and PD than controls in neurons (> 2% in both diseases), and for MSA also in non-neurons. In MSA substantia nigra (SN), we noted SNCA gains in > 3% of dopaminergic (DA) neurons (identified by neuromelanin) and neuromelanin-negative cells, including olig2-positive oligodendroglia. Cells with CNVs were more likely to have α-synuclein inclusions, in a pattern corresponding to cell categories mostly relevant to the disease: DA neurons in Lewy-body cases, and other cells in the striatonigral degeneration-dominant MSA variant (MSA-SND). Higher mosaicism levels in SN neuromelanin-negative cells may correlate with younger onset in typical MSA-SND, and in cingulate neurons with younger death in PD. Larger sample sizes will, however, be required to confirm these putative findings. We obtained genome-wide somatic CNV profiles from 169 cells from the substantia nigra of two MSA cases, and pons and putamen of one. These showed somatic CNVs in ~ 30% of cells, with clonality and origins in segmental duplications for some. CNVs had distinct profiles based on cell type, with neurons having a mix of gains and losses, and other cells having almost exclusively gains, although control data sets will be required to determine possible disease relevance. We propose that somatic SNCA CNVs may contribute to the aetiology and pathogenesis of synucleinopathies, and that genome-wide somatic CNVs in MSA brain merit further study.

Ovarian cancer is the most lethal gynecologic malignancy. About 75% of ovarian cancer patients relapse and/or develop chemo‐resistant disease after initial response to standard‐of‐care treatment with platinum‐based therapies. HER2 amplifications and overexpression in ovarian cancer are reported to vary, and responses to HER2 inhibitors have been poor. Next generation sequencing technologies in conjunction with testing using patient‐derived xenografts (PDX) allow validation of personalized treatments. Using a whole‐genome mate‐pair next generation sequencing (MPseq) protocol, we identified several high grade serous ovarian cancers (HGS‐OC) with DNA alterations in genes encoding members of the ERBB2 pathway. The efficiency of anti‐HER2 therapy was tested in three different PDX lines with the identified alterations and high levels of HER2 protein expression. Treatment responses to pertuzumab or pertuzumab/trastuzumab were compared in each PDX line WITH standard carboplatin and paclitaxel combination treatment. In all three PDX models, HER2‐targeted therapy resulted in significant inhibition of tumor growth compared with untreated controls. However, the responses in each case were inferior to those to chemotherapy, even for chemo‐resistant lines. When chemotherapy and HER2‐targeted therapy were administered together, a significant regression of tumor was observed after 6 weeks of treatment compared with chemotherapy alone. Post‐treatment analysis of these tissues revealed that inhibition of the ERBB2 pathway occurred at the level of phosphorylation and expression of downstream targets. In conclusion, while targeting of presumably activated ERBB2 pathway alone in HGS‐OC results in a modest treatment benefit, a combination therapy including both chemotherapy drugs and HER2 inhibitors provides a far better response. Further studies are needed to address development of recurrence and sensitivity of recurrent disease to HER2‐targeted therapy.

Abstract Objectives Inhibitors to the checkpoint proteins cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4) and programmed cell death protein 1 (PD‐1) are becoming widely used in cancer treatment. However, a lack of understanding of the patient response to treatment limits accurate identification of potential responders to immunotherapy. Methods In this study, we assessed the expression of PD‐1 and CTLA‐4 on 19 leucocyte populations in the peripheral blood of 74 cancer patients. A reference data set for PD‐1 and CTLA‐4 was established for 40 healthy volunteers to determine the normal expression patterns for these checkpoint proteins. Results Unsupervised hierarchical clustering found four immune profiles shared across the solid tumor types, while chronic lymphocytic leukaemia patients had an immune profile largely unique to them. Furthermore, we measured these leucocyte populations on an additional cohort of 16 cancer patients receiving the PD‐1 inhibitor pembrolizumab in order to identify differences between responders and non‐responders, as well as compared to healthy volunteers (n = 20). We observed that cancer patients had pre‐treatment PD‐1 and CTLA‐4 expression on their leucocyte populations at different levels compared to healthy volunteers and identified two leucocyte populations positive for CTLA‐4 that had not been previously described. We found higher levels of PD‐1+ CD3+ CD4− CD8− cells in patients with progressive disease and have identified it as a potential biomarker of response, as well as identifying other significant differences in phenotypes between responders and non‐responders. Conclusion These results are suggestive that categorisation of patients based on immune profiles may differentiate responders from non‐responders to immunotherapy for solid tumors.

Introduction There are no reliable blood biomarkers for monitoring endometrial cancer patients in the current clinical practice. Circulating tumor DNA (ctDNA) is emerging as a promising non-invasive method to measure tumor burden, define prognosis and monitor disease status in many solid cancers. In this pilot study, we investigated if unique tumor-specific DNA junctions can be used to detect ctDNA levels in patients with endometrial cancer. Methods Chromosomal rearrangements in primary tumors of eleven patients with high-grade or advanced stage endometrial cancer were determined by whole-genome Mate-Pair sequencing. Identified unique tumor-specific junctions were evaluated in pre- and six-week post-surgery patient plasma using individualized quantitative polymerase chain reaction (qPCR) assays. The relationship between clinicopathological features and detection of ctDNA was investigated. Results CtDNA was detected in 60% (6/10) of cases pre-surgery and in 27% (3/11) post-surgery. The detection of ctDNA pre-surgery was consistent with clinical indicators of aggressive disease such as advanced stage (80% - 4/5), lymphatic spread of disease (100% - 3/3), serous histology (80% - 4/5), deep myometrial invasion (100% - 3/3), lympho-vascular space invasion (75% - 3/4). All patients in which ctDNA was detected post-surgically had type II endometrial cancer. Discussion This pilot study demonstrates the feasibility of using personalized tumor-specific junction panels for detecting ctDNA in the plasma of endometrial cancer patients. Larger studies and longer follow-up are needed to validate the potential association between pre-surgical ctDNA detection and the presence of cancers with aggressive pathologic tumor characteristics or advanced stage observed in this study.

Current guidelines suggest screening all patients with idiopathic pulmonary arterial hypertension for genetic aberrations, particularly mutations in Bone Morphogenic Protein Receptor Type II ( BMPR2 ), the gene most commonly implicated in the pathogenesis of PAH. Herein, we present a novel technique used to identify a pathogenic germline BMPR2 alteration in a 36-year-old female and family members with hereditary pulmonary arterial hypertension who each screened negative by standard cytogenetics and molecular genetics testing.

FISH-based techniques to image and count mRNA in single cells can be limited by the photophysical properties of organic dyes. Here the authors develop photostable quantum dot FISH probes for multiplexed imaging.

Abstract Background HER2 positive (HER2+) breast cancers involve chromosomal structural alterations that act as oncogenic driver events. Methods We interrogated the genomic structure of 18 clinically-defined HER2+ breast tumors through integrated analysis of whole genome and transcriptome sequencing, coupled with clinical information. Results ERBB2 overexpression in 15 of these tumors was associated with ERBB2 amplification due to chromoanasynthesis with six of them containing single events and the other nine exhibiting multiple events. Two of the more complex cases had adverse clinical outcomes. Chromosomes 8 was commonly involved in the same chromoanasynthesis with 17. In ten cases where chromosome 8 was involved we observed NRG1 fusions (two cases), NRG1 amplification (one case), FGFR1 amplification and ADAM32 or ADAM5 fusions. ERBB3 over-expression was associated with NRG1 fusions and EGFR and ERBB3 expressions were anti-correlated. Of the remaining three cases, one had a small duplication fully encompassing ERBB2 and was accompanied with a pathogenic mutation. Conclusion Chromoanasynthesis involving chromosome 17 can lead to ERBB2 amplifications in HER2+ breast cancer. However, additional large genomic alterations contribute to a high level of genomic complexity, generating the hypothesis that worse outcome could be associated with multiple chromoanasynthetic events.

Abstract Metalloproteinases (MMPs) contribute to tissue remodeling and acute inflammation not only by degrading extracellular matrix proteins but also by controlling the influx of chemokines through the regulation and shedding of syndecans. B-lymphocytes, in addition to their well-known function as antibody producing cells, participate in the innate immune response by secreting inflammatory cytokines and chemokines. However, there is little information about the role of B-lymphocytes in the regulation of MMPs; consequently, herein we investigated whether activated human circulating B-lymphocytes contributed to the secretion of MMPs. We demonstrate that B-lymphocytes activated by un-methylated CpG motifs, found in bacterial DNA, and β-glucans, found in the cell wall of fungi, both induced MMP-7. Interestingly, while CpG-stimulated cells activated the mTOR pathway via TLR9 receptor to induced MMP-7, β-glucan-stimulated cells were mTOR-independent and used Dectin-1 receptor. B-lymphocytes did not seem to have a major role in the secretion of tissue inhibitors of metalloproteinases (TIMPs). However, secreted MMP-7 participated in the shedding of Syndecan-4 from the surface of B-lymphocytes. In conclusion, circulating human B-lymphocytes contribute to the regulation of the innate immune system by participating in the secretion of MMP-7 which in turn is important for the shedding of Syndecan-4 in response to infectious stimuli.

Overexpression of TOP2A is associated with risk of systemic progression in prostate cancer patients, and higher levels of TOP2A were found in hormone-resistant cases. To elucidate the mechanism by which high levels of TOP2A contribute to tumor progression we generated TOP2A overexpressing prostate cancer cell lines. We show that TOP2A promotes tumor aggressiveness by inducing chromosomal rearrangements of genes that contribute to a more invasive phenotype. Anti-androgen treatment alone was ineffective in killing TOP2A overexpressing cells due to activation of an androgen receptor network. TOP2A poisons killed tumor cells more efficiently early in the progression course, while at later stages they provided greater benefit when combined with anti-androgen therapy. Mechanistically, we find that TOP2A enhances androgen signaling by facilitating transcription of androgen responsive genes, thereby promoting tumor cell growth. These studies revealed a relationship between TOP2A and androgen receptor signaling pathway that contributes to prostate cancer progression and confers sensitivity to treatments.

Hypoxia has been associated with malignant progression, metastasis and resistance to therapy. Hence, we studied expression of hypoxia-regulated genes in 100 prostate cancer (CaP) bulk tissues and 71 adjacent benign tissues. We found 24 transcripts significantly overexpressed (p ≤ 0.02). Importantly, higher transcript levels of disc large (drosophila) homolog-associated protein 5 (DLGAP5)/discs large homolog 7 (DLG7)/hepatoma up-regulated protein (HURP), hyaluronan-mediated motility receptor (HMMR) and cyclin B1 (CCNB1) were associated with higher Gleason score and more advanced systemic progression. Since the products of HMMR and CCNB1 have been identified recently as molecular markers of CaP progression, we postulated that DLG7 has prognostic value too. To test this hypothesis, we measured transcript levels for DLG7 in a 150-pair case-control cohort. The cases (progression to systemic disease within six years of surgery) and controls (no progression within eight years) were matched for clinical and pathologic prognostic variables, including grade, stage, and preoperative serum levels of PSA. The overall prognostic ability of DLG7, as tested in receiver operating characteristic analysis was of 0.74 (95% CI, 0.68 to 0.8). Overall, our data indicate that expression of DLG7, a hypoxia-controlled gene, holds prognostic potential in high-risk CaP; this also demonstrates that variation of oxygen tension may constitute a tool for identification of novel biomarkers for CaP.