Single-cell RNA sequencing (scRNA-seq) is a powerful tool for studying complex biological systems, such as tumor heterogeneity and tissue microenvironments. However, the sources of technical and ...biological variation in primary solid tumor tissues and patient-derived mouse xenografts for scRNA-seq are not well understood.
We use low temperature (6 °C) protease and collagenase (37 °C) to identify the transcriptional signatures associated with tissue dissociation across a diverse scRNA-seq dataset comprising 155,165 cells from patient cancer tissues, patient-derived breast cancer xenografts, and cancer cell lines. We observe substantial variation in standard quality control metrics of cell viability across conditions and tissues. From the contrast between tissue protease dissociation at 37 °C or 6 °C, we observe that collagenase digestion results in a stress response. We derive a core gene set of 512 heat shock and stress response genes, including FOS and JUN, induced by collagenase (37 °C), which are minimized by dissociation with a cold active protease (6 °C). While induction of these genes was highly conserved across all cell types, cell type-specific responses to collagenase digestion were observed in patient tissues.
The method and conditions of tumor dissociation influence cell yield and transcriptome state and are both tissue- and cell-type dependent. Interpretation of stress pathway expression differences in cancer single-cell studies, including components of surface immune recognition such as MHC class I, may be especially confounded. We define a core set of 512 genes that can assist with the identification of such effects in dissociated scRNA-seq experiments.
Taq DNA polymerase functions at elevated temperatures with fast conformational dynamics-regimes previously inaccessible to mechanistic, single-molecule studies. Here, single-walled carbon nanotube ...transistors recorded the motions of Taq molecules processing matched or mismatched template-deoxynucleotide triphosphate pairs from 22° to 85°C. By using four enzyme orientations, the whole-enzyme closures of nucleotide incorporations were distinguished from more rapid, 20-μs closures of Taq's fingers domain testing complementarity and orientation. On average, one transient closure was observed for every nucleotide binding event; even complementary substrate pairs averaged five transient closures between each catalytic incorporation at 72°C. The rate and duration of the transient closures and the catalytic events had almost no temperature dependence, leaving all of Taq's temperature sensitivity to its rate-determining open state.
Stroke can result in significant mental and physical impairment. Training health care professionals on effective strategies for mitigating stroke-related quality-of-life issues is crucial in ...facilitating comprehensive stroke management. This study aimed to evaluate the impact of an interprofessional education (IPE) experience on students’ attitudes regarding poststroke disability.
In this pre-post interventional study, pharmacy and medical students received an electronic patient chart and a store-and-forward video depicting physical and cognitive impairment in a patient with stroke. Students were instructed to discuss the acute management and postdischarge needs of the patient from an advocacy perspective. After the IPE experience, students completed the Student Perceptions of Interprofessional Clinical Education-Revised, version 2 and an unvalidated disability attitudes survey. The surveys were analyzed using a paired t test. In addition, students reflected on the prompt, “What are some things you had NOT considered prior to this IPE?”
A total of 708 students completed the surveys. After IPE, there was a significant improvement in all domains of the Student Perceptions of Interprofessional Clinical Education-Revised, version 2. On the disability survey, there was significant improvement on all statements, including “rate your comfort with…”: “discussing the expected disabilities associated with new-onset stroke” and “discussing strategies for improving the quality of life of a patient who has long-term disabilities.” On the self-reflections, 31.7% (n = 211) had not considered the need for poststroke care services before this IPE.
This IPE experience was instrumental in improving student perspectives regarding poststroke disability.
Abstract
Introduction Breast cancer is the most common cancer in female and triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer which shows high rate of recurrence ...and metastasis. Malignant cells that comprise primary tumor are heterogeneous and during disease progression selection of tumor cells occur as a mean to adapt and survive. Tumor heterogeneity can be studied by grouping cells as clones which refer to a group of cells related to each other by descent from a unitary origin. Understanding the mechanism of clonal dynamics and selection during cancer evolution is important to develop new therapeutic strategies for cancer metastasis. Moreover, it is imperative to be able to detect rare subclones that are responsible for metastasis. Thus, single-cell analysis is critical to identify cellular heterogeneity of cancer and molecular basis of metastatic phenotype in depth. Measuring genome and transcriptome in single-cell level will enable us to discover clonal dynamics during cancer metastasis and infer molecular determinants of metastasis fitness. Here, we propose to investigate the clonal dynamics, genomic, and transcriptomic profiles of human breast tumor metastasis using TNBC patient-derived xenograft (PDX) model to understand the mechanism of breast cancer progression and metastasis. Methods Tumor cells from previously established PDXs were transplanted into mammary fat pad of mice. Tumors were removed when it reached maximum allowed endpoint size (1,000mm3) and mice were monitored and allowed to grow metastasis. PDXs that developed primary tumor and metastasis were selected for subsequent single cell analysis. Single-cell whole-genome sequencing (scWGS) was performed using direct library preparation (DLP+) method which is preamplification-free single-cell genome sequencing approach. scWGS data was used to call cell-specific copy number events, which allows us to cluster cells and identify clones. Hierarchical clustering was used to identify clonal structure of each sample. Phylogenetic tree was computed using copy number data of samples from each PDX. Results Nine different TNBC PDX lines were tested and 6 PDX lines developed metastasis. We focused on 2 PDX lines of which metastatic tissues were available for single cell sequencing. SA919 developed metastatic mass near cervical or lumbar spine area. SA535 developed metastasis to lung, axillary or inguinal area and tumor recurrence at mammary fat pad site. scWGS for SA919 and SA535 was carried out and we generated libraries from single cells of primary tumor and its metastases. Hierarchical clustering using copy number data revealed clonal structure of primary tumor and metastases. SA919 showed oligoclonal primary tumor and metastasis whereas SA535 showed polyclonal metastasis from polyclonal primary tumor. scWGS data of primary tumor and its metastases in each PDX were merged to compute phylogenetic trees. Phylogenetic analysis revealed that not all clones of primary tumor contributed to metastasis in both PDXs. Different metastatic lesions of SA535 harbored different clones from primary tumor. For example, clones consisting left axillary metastasis and right axillary metastasis in SA535 were different from each other, however they both originated from primary tumor clones. Metastatic lesions from SA919 and SA535 also had newly appearing clones that were not present in primary tumor inferring clonal evolution during metastasis. Conclusion We were able to capture different patterns of metastasis in several PDXs. Phylogenetic analysis using scWGS data revealed clonal relationship between primary tumor and metastases. Further analysis of single cell genomic and transcriptomic profiles will provide deeper understanding of metastasis in breast cancer.
Citation Format: Hakwoo Lee, Farhia Kabeer, Ciara O'Flanagan, Jazmine Brimhall, Justina Biele, Biexi Wang, Teresa Ruiz Algara, So Ra Lee, Daniel Lai, Michael Yuen, Simong Song, Patricia Ye, Jenifer Pham, Richard Moore, Andy J Mungall, Sohrab P Shah, Samuel Aparicio. Single-cell analysis of breast cancer metastasis using patient-derived xenograft model reveals clonal relationship between primary tumor and its metastases abstract. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD8-05.
Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNA), has been impeded by lack of time series single cell sampling of polyclonal ...populations and temporal statistical models
1
–
7
. Here, we generated 42,000 genomes from multi-year time series single cell whole genome sequencing (scWGS) of breast epithelium and primary triple negative breast cancer (TNBC) patient derived xenografts (PDX), revealing the nature of CNA defined clonal fitness dynamics induced by
TP53
mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model
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,
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to infer clonal fitness, we found that
TP53
mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in
TP53
mutant TNBC PDX models, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDX resulted in cisplatin resistant clones emerging from low fitness phylogenetic lineages in the untreated setting. Conversely, high fitness clones from treatment naive controls were eradicated, signaling an inversion of the fitness landscape. Finally, upon release of drug selective pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Taken together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours.
Fifty percent of lung adenocarcinomas harbor somatic mutations in six genes that encode proteins in the EGFR signaling pathway, i.e., EGFR, HER2/ERBB2, HER4/ERBB4, PIK3CA, BRAF, and KRAS. We ...performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this signaling pathway that could contribute to lung tumorigenesis.
We analyzed genomic DNA from a total of 261 resected, clinically annotated non-small cell lung cancer (NSCLC) specimens. The coding sequences of 39 genes were screened for somatic mutations via high-throughput dideoxynucleotide sequencing of PCR-amplified gene products. Mutations were considered to be somatic only if they were found in an independent tumor-derived PCR product but not in matched normal tissue. Sequencing of 9MB of tumor sequence identified 239 putative genetic variants. We further examined 22 variants found in RAS family genes and 135 variants localized to exons encoding the kinase domain of respective proteins. We identified a total of 37 non-synonymous somatic mutations; 36 were found collectively in EGFR, KRAS, BRAF, and PIK3CA. One somatic mutation was a previously unreported mutation in the kinase domain (exon 16) of FGFR4 (Glu681Lys), identified in 1 of 158 tumors. The FGFR4 mutation is analogous to a reported tumor-specific somatic mutation in ERBB2 and is located in the same exon as a previously reported kinase domain mutation in FGFR4 (Pro712Thr) in a lung adenocarcinoma cell line.
This study is one of the first comprehensive mutational analyses of major genes in a specific signaling pathway in a sizeable cohort of lung adenocarcinomas. Our results suggest the majority of gain-of-function mutations within kinase genes in the EGFR signaling pathway have already been identified. Our findings also implicate FGFR4 in the pathogenesis of a subset of lung adenocarcinomas.
Purpose
To evaluate the time course of improvements in clinical convergence measures for children with symptomatic convergence insufficiency treated with office‐based vergence/accommodative therapy.
...Methods
We evaluated convergence measures from 205, 9‐ to 14‐year‐old children with symptomatic convergence insufficiency randomised to office‐based vergence/accommodative therapy in the Convergence Insufficiency Treatment Trial – Attention and Reading Trial (CITT‐ART). Near‐point of convergence (NPC) and near‐positive fusional vergence (PFV) were measured at baseline and after 4, 8, 12 and 16 weeks of therapy; mean change in NPC and PFV between these time points were compared using repeated measures analysis of variance. Rates of change in NPC and PFV from: (1) baseline to 4 weeks and (2) 4–16 weeks were calculated. For each time point, the proportion of participants to first meet the normal criterion for NPC (<6 cm), PFV blur (break if no blur; >15Δ and >2 times the exodeviation) and convergence composite (NPC and PFV both normal) were calculated.
Results
The greatest change in NPC and PFV (7.6 cm and 12.7 Δ) and the fastest rate of improvement in NPC and PFV (1.9 cm/week and 3.2 Δ/week, respectively) were both found during the first 4 weeks of therapy, with both slowing over the subsequent 12 weeks. After 12 weeks of therapy, the NPC, PFV and convergence composite were normal in 93.2%, 91.7% and 87.8% of participants, respectively, and normalised with another 4 weeks of therapy in 4.4%, 2.0% and 4.4% of participants, respectively.
Conclusion
Although the greatest improvements in NPC and PFV occurred in the first 4 weeks of therapy, most participants had weekly improvements over the subsequent 12 weeks of treatment. While most children with convergence insufficiency obtained normal convergence following 12 weeks of therapy, an additional 4 weeks of vergence/accommodative therapy may be beneficial for some participants.
Purpose
To determine the effectiveness of office‐based vergence/accommodative therapy for improving accommodative amplitude and accommodative facility in children with symptomatic convergence ...insufficiency and accommodative dysfunction.
Methods
We report changes in accommodative function following therapy among participants in the Convergence Insufficiency Treatment Trial – Attention and Reading Trial with decreased accommodative amplitude (115 participants in vergence/accommodative therapy; 65 in placebo therapy) or decreased accommodative facility (71 participants in vergence/accommodative therapy; 37 in placebo therapy) at baseline. The primary analysis compared mean change in amplitude and facility between the vergence/accommodative and placebo therapy groups using analyses of variance models after 4, 8, 12 and 16 weeks of treatment. The proportions of participants with normal amplitude and facility at each time point were calculated. The average rate of change in amplitude and facility from baseline to week 4, and from weeks 4 to 16, were determined in the vergence/accommodative therapy group.
Results
From baseline to 16 weeks, the mean improvement in amplitude was 8.6 dioptres (D) and 5.2 D in the vergence/accommodative and placebo therapy groups, respectively (mean difference = 3.5 D, 95% confidence interval (CI): 1.5 to 5.5 D; p = 0.01). The mean improvement in facility was 13.5 cycles per minute (cpm) and 7.6 cpm in the vergence/accommodative and placebo therapy groups, respectively (mean difference = 5.8 cpm, 95% CI: 3.8 to 7.9 cpm; p < 0.0001). Significantly greater proportions of participants treated with vergence/accommodative therapy achieved a normal amplitude (69% vs. 32%, difference = 37%, 95% CI: 22 to 51%; p < 0.0001) and facility (85% vs. 49%, difference = 36%, 95% CI: 18 to 55%; p < 0.0001) than those who received placebo therapy. In the vergence/accommodative therapy group, amplitude increased at an average rate of 1.5 D per week during the first 4 weeks (p < 0.0001), then slowed to 0.2 D per week (p = 0.002) from weeks 4 to 16. Similarly, facility increased at an average rate of 1.5 cpm per week during the first 4 weeks (p < 0.0001), then slowed to 0.6 cpm per week from weeks 4 to 16 (p < 0.0001).
Conclusion
Office‐based vergence/accommodative therapy is effective for improving accommodative function in children with symptomatic convergence insufficiency and coexisting accommodative dysfunction.
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